Novel Mechanism of β0 Thalassemia: Missense Mutation of the Last Nucleotide of β Globin Exon 1 (G->C) Leads to Undetectable Transcript and Mutant Peptide.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1598-1598
Author(s):  
Neeraj Agarwal ◽  
Mariluz P. Mojica-Henshaw ◽  
Ferdane Kutlar ◽  
Amos Gaikwad ◽  
Ching N. Ou ◽  
...  
Keyword(s):  
African American ◽  
Missense Mutation ◽  
Sickle Cell ◽  
Globin Gene ◽  
Enzyme Analysis ◽  
African American Patient ◽  
Globin Mrna ◽  
Mutant Peptide ◽  
Exon 1

Abstract Hemoglobin Monroe (Hb Monroe) results from a point mutation (G->C) in the last nucleotide of β globin exon 1, which is also the penultimate nucleotide of codon 30 of the β globin mRNA (AGG->ACG/Arg->Thr). Hb Monroe was described seventeen years ago simultaneously by two groups: in an African American female with β thalassemia intermedia, wherein the thalassemia was thought to result from highly unstable peptide (Hemoglobin.1989;13:67); and in a North African female with compound β thalassemia (Proc Natl Acad Sci U S A.1989;86:1041) wherein the Hb Monroe mutation was thought to result in abnormal pre-mRNA splicing as detected in an in vitro cell-free transcription assay. We evaluated a 31-year old previously asymptomatic woman of Asian Indian (Bengali) descent, who presented with flu like symptoms and found to have low hemoglobin level (9.5 gm/dL), microcytosis (MCV 68 fL), moderately elevated liver enzymes and serum ferritin concentration of 3000 ng/ml. A liver biopsy revealed increased liver iron and significant fibrosis. Hemoglobin analysis, which was interpreted as compound heterozygosity for HbE/β0 thalassemia, revealed HbF: 51%, HbE: 43.2%, HbA2: 5.8%. β globin gene sequencing showed Hb Monroe and E mutations. The asymptomatic brother of the proband had borderline anemia (Hb 12 gm %), microcytosis (MCV 70 fl), HbF: 8.5 %, HbA: 87.2%, HbA2: 5.0% and was heterozygous for Hb Monroe mutation by Bme 15801 restriction enzyme analysis of genomic DNA. We set out to determine the molecular basis of the thalassemia phenotype associated with Hb Monroe mutation and whether this mutation in our subjects is present on African haplotype or had arisen independently. Since we could not detect the mutant peptide either in fresh hemolysate or reticulocyte enriched preparations; we expanded the peripheral blood erythroid progenitor cells in vitro of both proband and her brother. Hemoglobin analysis by both HPLC and mass spectrophotometry did not detect Hb Monroe peptide in the expanded cells. β globin cDNA from the reticulocytes and expanded erythroid progenitors was amplified using three different primer sets and no splice variants were detectable. Sequencing of the amplified cDNA revealed only normal β globin mRNA transcript. Other β globin gene mutations cis to Hb Monroe are being ruled out; to date, we have not found any promoter region or stop codon mutations, deletions or splicing mutations from promoter - 90 region to 3′ UTR including poly-A region. Haplotype analysis revealed a different haplotype from the two African American patients, indicating an independent origin of Hb Monroe mutation in our cases. Interestingly, both of our cases have elevated HbF, as did the two originally reported Hb Monroe patients (16.5 and 85%) and a currently unreported African American patient with sickle cell - β0 thalassemia due to Hb Monroe (11.4%). Hb F was also high in a group of nine patients reported with sickle cell - β0 thalassemia due to Hb Monroe (3.1%– 8.9%; Hemoglobin.1998; 22:153). We conclude that this missense mutation, IVS1-1 (G->C/Arg 30 Thr) results in undetectable transcript and mutant Hb peptide leading to β0 thalassemia. The molecular mechanism of the undetectable mutant transcript is being investigated.

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 Missense mutation of the last nucleotide of exon 1 (G->C) of globin gene not only leads to undetectable mutant peptide and transcript but also interferes with the expression of wild allele

Haematologica ◽  
2007 ◽  
Vol 92 (12) ◽  
pp. 1715-1716 ◽  
Author(s):  
N. Agarwal ◽  
F. Kutlar ◽  
M. P. Mojica-Henshaw ◽  
C. N. Ou ◽  
A. Gaikwad ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Globin Gene ◽  
Mutant Peptide ◽  
Wild Allele ◽  
Exon 1

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 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.

Hypoxia-induced acute lung injury in murine models of sickle cell disease

2004 ◽  
Vol 286 (4) ◽  
pp. L705-L714 ◽  
Author(s):  
Kirkwood A. Pritchard ◽  
Jingsong Ou ◽  
Zhijun Ou ◽  
Yang Shi ◽  
James P. Franciosi ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Lung Injury ◽  
Sickle Cell ◽  
Globin Gene ◽  
Heat Shock Protein 90 ◽  
Cell Disease ◽  
Vascular Congestion ◽  
Nitrite Nitrate ◽  
Endothelial Nitric Oxide

Vaso-occlusive events are the major source of morbidity and mortality in sickle cell disease (SCD); however, the pathogenic mechanisms driving these events remain unclear. Using hypoxia to induce pulmonary injury, we investigated mechanisms by which sickle hemoglobin increases susceptibility to lung injury in a murine model of SCD, where mice either exclusively express the human α/sickle β-globin (hαβS) transgene (SCD mice) or are heterozygous for the normal murine β-globin gene and express the hαβStransgene (mβ+/-, hαβS+/-; heterozygote SCD mice). Under normoxia, lungs from the SCD mice contained higher levels of xanthine oxidase (XO), nitrotyrosine, and cGMP than controls (C57BL/6 mice). Hypoxia increased XO and nitrotyrosine and decreased cGMP content in the lungs of all mice. After hypoxia, vascular congestion was increased in lungs with a greater content of XO and nitrotyrosine. Under normoxia, the association of heat shock protein 90 (HSP90) with endothelial nitric oxide synthase (eNOS) in lungs of SCD and heterozygote SCD mice was decreased compared with the levels of association in lungs of controls. Hypoxia further decreased association of HSP90 with eNOS in lungs of SCD and heterozygote SCD mice, but not in the control lungs. Pretreatment of rat pulmonary microvascular endothelial cells in vitro with xanthine/XO decreased A-23187-stimulated nitrite + nitrate production and HSP90 interactions with eNOS. These data support the hypotheses that hypoxia increases XO release from ischemic tissues and that the local increase in XO-induced oxidative stress can then inhibit HSP90 interactions with eNOS, decreasing ·NO generation and predisposing the lung to vaso-occlusion.

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 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.

Aroyl-Pyrrolyl-Hydroxy-Amides (APHAs), a Novel Family of Synthetic Histone Deacetylases Inhibitors, Are Potent Inducers of Human g-Globin Gene Expression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1216-1216
Author(s):  
Antonello Mai ◽  
Silvio Massa ◽  
Antonella Di Noia ◽  
Katija Jelicic ◽  
Elena Alfani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Histone Deacetylases ◽  
Globin Gene ◽  
Renilla Luciferase ◽  
In Vitro Assays ◽  
Luciferase Reporter ◽  
Globin Gene Expression

Abstract Post-natal pharmacological reactivation of HbF, by restoring the unbalanced α/non-α globin chain production in red cells of patients affected by β-thalassemia or sickle cell anemia, represents a potential cure for these diseases. Many classes of compounds have been identified capable to induce Hb F synthesis in vitro by acting at different levels of the globin gene expression regulatory machinery. One of these classes is represented by inhibitors of a family of enzymes, the histone deacetylases (HDACs), involved in chromatin remodelling and gene transcription regulation. HDACs act in multi-protein complexes that remove acetyl groups from lysine residues on several proteins, including histones and are divided into three distinct structural classes, depending on whether their catalytic activity is zinc (class I/II)- or NAD+ (class III)-dependent. The effects of the HDACs inhibitors identified so far on HbF synthesis is, however, modest and often associated with high toxicity. Therefore, the potential of their clinical use is unclear. We have recently described a new family of synthetic HDACs inhibitors, the Aroyl-pyrrolyl-hydroxy-amides (APHAs), that induce differentiation, growth arrest and/or apoptosis of transformed cell in culture [Mai A et al, J Med Chem2004;47:1098]. In this study, we investigate the capability of 10 different APHA compounds to induce Hb F in two in vitro assays. One assay is based on the ability of APHA compounds to activate either the human Aγ-driven Firefly (Aγ-F) or the β-promoter drives Renilla Luciferase (β-R) reporter in GM979 cells stably transfected with a Dual Luciferase Reporter construct. The second assay is represented by the induction of γ-globin expression (by quantitative RT-PCR) in primary adult erythroblasts obtained in HEMA cultures of mononuclear cells from normal donors. The majority of the compounds tested did not significantly increased the Aγ−F (Aγ−F+β−R) reporter ratio in GM979 cells. However, the compound MC1575 increased by 3-fold (from 0.09 to 0.30) the reporter ratio in GM979 cells at a concentration of 20 μM, with modest effects of the proliferation activity of GM979 cells over the three days of the assay. When MC1575 was added at a concentration of 2–10 μM in cultures of primary adult erythroblasts induced to differentiate in serum-free media for 4 days, it induced a three fold increase of the γ/(γ+β) globin ratio (from 0.04 to 0.12), with no apparent cellular toxicity. Among the HDAC inhibitors tested in this study, MC1575 was not the most potent inhibitor of total enzyme activity. However, it was the compound that most selectively inhibited the activity of the maize homologue of mammalian class IIa HDAC enzymes [Mai et al, J Med Chem2003;46:4826]. These results are consistent with the hypothesis that each class of histone deacetylases might have a specific biological function and indicate that those of class IIa might represent the enzymes most specifically involved in globin gene regulation. We suggest that, by targeting the chemical inhibitors toward the catalytic domain of this class of enzymes, it should be possible to identify more specific, more potent and less toxic compounds for pharmacological treatment of β-thalassemia or sickle cell anemia.

Decreased Rate of β-GlobinL1 Allele Transcription Due to Intronic LINE-1 Insertion In the β-Globin gene Is Associated with β-globinL1 Promoter and Enhancer Hypermethylation Which Is Not Reverted by Decitabine

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2073-2073
Author(s):  
Lucie Piterkova ◽  
Jana Kucerova ◽  
Karel Indrak ◽  
Vladimir Divoky
Keyword(s):  
Internal Control ◽  
Promoter Region ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Methylation Status ◽  
In Vitro Transcription ◽  
Regulatory Sequence ◽  
Globin Mrna ◽  
Enhancer Region

Abstract Abstract 2073 We have previously described a unique etiology of β-thalassemia due retrotransposon LINE-1 (L1) insertion into the intron-2 of the β-globin gene in a mother and daughter of Ukrainian descent who exhibited typical laboratory features of β-thalassemia trait (1, 2). The affected β-globinL1 gene generated roughly 10–15% of the total β−globin message (1). In order to unravel how the intronic insertion of transposable element attenuates the β-globin expression, interspecific hybrids of the propositus′ lymphocytes and mouse erythroleukemia (MEL) cells were generated (laboratory of Dr. T. Papayannopoulou, Seattle, WA) and used for expression studies. The total RNA from differently treated (emetine, 5-aza-2′-deoxycytidine (Sigma)) in vitro cultured cell hybrids were isolated and β-globin transcripts were analyzed by Real-Time qPCR with gene-specific primers. Nuclei from hybrid MEL cells were isolated to performed nuclear run-on assay. Bisulfite modification was done on genomic DNA from MEL hybrids and the promoter and enhancer regions of β-globin gene were amplified, PCR products were subcloned and sequenced. We demonstrated that the observed reduction in steady-state level of β-globin mRNA is partially caused by aberrant splicing followed by activation of nonsense-mediated decay (NMD) pathway, leading to increased degradation of aberrant β-globin mRNA variants. Reduction in expression of β-globin mRNA from β-globinL1 allele comes also from altered rate of transcription. We performed PCR-based nuclear run-on assay and forty minutes of in vitro transcription revealed 30% decrease in β-globinL1 allele transcription rate compared to wild-type β-globin allele. It is known that L1 regulatory regions are highly methylated (3) but there is no evidence so far demonstrating retrotransposon-mediated epigenetic control of neighboring genes. Therefore we determined the methylation status of 6 CpGs between −415 bp and +110 bp (bases upstream and downstream of transcription start) on the promoter region and 3 CpGs between +442 bp to +592 bp on the enhancer region (bases downstream of the poly(A) signal) of the mutated and normal β−globin alleles. Enhancer region of the mutated allele was 100% methylated (100% Me-CpG versus 9% Me-CpG in control), and promoter region was partially methylated (≈ 65% Me−CpG versus 35% Me-CpG in control). Surprisingly, treatment of induced hybrid MEL cells with demethylation agent (5-aza-2′-deoxycytidine, decitabine) did not change the methylation profile and consequently did not increase the expression of β-globinL1 mRNA whereas the expression of γ-globin gene, which was used as an internal control, was increased. We therefore hypothesize that decreased rate of transcription from β-globinL1 allele is associated with altered DNA topology caused by the L1 insertion and β-globinL1 promoter-enhancer displacement. Resulting chromatin modifications lead to permanent β-globinL1 gene silencing and hypermethylation of regulatory sequence refractory to demethylating agent. Divoky V et al: Blood. 1996;88 (suppl1):148a. Kimberland ML, Divoky V et al: Hum Mol Genet. 1999;8:1557-1560. Hata K, Sakaki Y: Gene. 1997;189:227-234. Grant support: NS10281 −3/2009 Ministry of Health, Czech Republic and Internal grant agency of Palacky University grant No. LF_2010_013. Disclosures: No relevant conflicts of interest to declare.

Selective Inhibitors of Arginine Methyl Transferase 5 (PRMT5) As a Novel Treatment for β-Thalassemia and Sickle Cell Disease.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2129-2129 ◽  
Author(s):  
Ian Street ◽  
Brendon Monahan ◽  
Hendrik Falk ◽  
Elizabeth Allan ◽  
Ylva Bergman ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Cell Disease ◽  
Globin Mrna ◽  
Cpg Dinucleotides ◽  
Radiometric Assay ◽  
Adult Bone

Abstract Abstract 2129 The developmental switch in human β-like globin gene subtype from fetal (γ) to adult (β) that begins at birth foreshadows the onset of the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD). In the clinical setting it is established that β-thalassemia and SCD patients with hereditary persistence of fetal hemoglobin mutations enjoy a significant amelioration of disease severity due to the continued expression of γ-globin. This has prompted the search for therapeutic strategies to reverse γ-globin gene silencing. Central to the mechanism of γ-gene silencing is DNA methylation, which marks critical CpG dinucleotides flanking the γ-gene transcriptional initiation site in adult bone marrow erythroid cells. These marks are established by recruitment of DNMT3A, a DNA methyltransferase, to the γ-globin promoter by protein arginine methyltransferase 5 (PRMT5)[Zhao Q et al. Nat Struct Mol Biol. 2009;16(3):304–311]. PRMT5 catalyses the symmetric dimethylation of arginine 3 of Histone 4 (H4R3me2), which serves as a template for direct binding of DNMT3A and the subsequent DNA methylation of the γ-gene promoter. Loss of PRMT5 or its enzymatic activity is sufficient to induce demethylation of the CpG dinucleotides and reactivation of γ-globin gene expression [Rank, G., et al. Blood, 116(9), 1585–92]. Based on these observations we hypothesize that small molecule inhibitors of PRMT5 activity could provide a beneficial treatment for β-thalassemia and SCD. To identify small molecule inhibitors of PRMT5 a high throughput screen (HTS) was performed. Both radiometric and non-radiometric assay formats were developed to support the screening campaign. The radiometric assay format measures the ability of PRMT5 purified from K562 cells to catalyse the labelling of a short peptide based on the N-terminal sequence of Histone H4 by 3H-Methyl-S-Adenosyl-L-methionine (SAM). In contrast, the non-radiometric assay format employs recombinant PRMT5/MEP50 and measures the production of S-adenosyl-L-homocysteine (SAH), which is generated by PRMT5-catalysed methylation of H4 peptide. SAH is measured with Transcreener EPIGEN” and the assay is formatted in 1536-well microtitre plates in a total assay volume of 4 μL. Using these assays, a chemical library of 350,000 lead-like molecules and known pharmacologically active agents was screened to identify inhibitors of PRMT5 methyltransferase activity. A number of compounds with low micromolar or submicromolar inhibitory activity were identified by the HTS campaign, and six were selected for re-synthesis. The inhibitory activity of five of the six compounds was confirmed. To provide an initial appraisal of inhibitor selectivity the five active compounds were subsequently tested against a panel of enzymes consisting of 23 protein and DNA methyltransferases and 12 kinases. These compounds were found to be remarkably selective PRMT5 inhibitors, inhibition of MLL4 being the only significant off-target activity noted for one of the scaffolds. We have established a critical path for selection and progression of new chemical analogues which entails testing the compounds for: i) inhibition of PRMT5, other protein methyl transferases and kinases; ii) the ability to induce expression of γ-globin mRNA in the K562 erythroleukemic cell line; iii) the ability to induce expression of γ-globin mRNA in adult bone marrow erythroid cells; and iv) the induction of γ-globin in vivo in β-YAC mice, a transgenic model which carries the 250-kb human globin locus. In parallel, the physicochemical, metabolism, and pharmacokinetic properties of the most promising compounds are also determined. Medicinal chemistry efforts have now produced molecules with > 100-fold increased inhibitory potency against PRMT5 compared to the original hits, and preliminary results indicate that the more potent compounds have the ability to induce γ-globin mRNA in our cell based models. These early results illustrate the potential of PRMT5 inhibitors as a novel approach for the treatment of β-thalassemia and sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

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