Pumilio1 (PUM1) Expression, Sickle Cell Anemia, β-thalassemia Intermedia

Study Overview
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Brief Summary
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To study the expression pattern of PUM1 gene in patients with sickle cell anemia and β-thalassemia intermedia. To detect PUM1 protein levels in sickle cell anemia and β-thalassemia intermedia patients. To correlate PUM1 gene expression pattern and protein levels with HbF levels in sickle cell anemia and β-thalassemia intermedia patients.

Detailed Description
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The disorders of β-hemoglobin, sickle cell disease (SCD) and β-thalassemia, are major causes of morbidity and mortality worldwide. These diseases are the most common genetic disorders in the world. SCD is due to a single base-pair point mutation in the β-globin gene resulting in the substitution of valine for glutamic acid in the β-globin chain. The pathophysiology is directly related to polymerization of deoxygenated hemoglobin, leading to a cascade of pathologic events including erythrocyte sickling, vaso-occlusion, and hemolysis. In β-thalassemia, insufficient production of the β-globin molecule results in an excess of free α-globin chains that can precipitate within erythroid precursors, impairing their maturation and leads to death of these precursors and ineffective production of erythroid cells. As a result, a significant anemia occurs and the consequent expansion of erythroid precursors can lead to secondary problems in bones and other organs. The hemoglobin molecule is a tetramer composed of two subunits of α-like globin peptides and two subunits of the β-like globin peptides, along with heme moieties. β-globin switching from fetal γ-globin (HBG1 and HBG2) to adult β-globin is a developmental process that occurs in erythrocytes at around the time of birth. Fetal hemoglobin (HbF) induction in adult erythrocytes is an effective therapeutic strategy for SCD and β-thalassemia. Pumilio1 (PUM1) is a novel target of the erythroid master transcription factor erythroid Krüppel-like factor (EKLF). PUM1 is a member of Pumilio-Fem3-binding factor (PUF) family of sequence specific RNA-binding proteins, acts as a posttranscriptional repressor by binding to the 3' untranslated region (3'-UTR) of messenger RNA (mRNA). It peaks during terminal erythroid differentiation and binds to fetal γ-globin (HBG1) mRNA and impairs its stability and translation. HBG1 has 2 core PUM1 consensus binding sites, but HBG2 and adult globins do not. Knockdown of PUM1 leads to a robust increase in HBF (∼22%) without affecting β-globin levels in human erythroid cells. Moreover, targeting PUM1 does not affect erythropoiesis, which provides a potentially safe and effective therapeutic strategy for SCD and β-thalassemia. Also it was found that elevated HbF levels in the absence of anemia in an individual with a novel heterozygous PUM1 mutation in the RNA-binding domain, which suggests that PUM1 is a critical player during human hemoglobin switching.

Official Title
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Detection of RNA Binding Protein Pumilio1 (PUM1) Expression in Patients With Sickle Cell Anemia and β-thalassemia Intermedia

Conditions
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Sickle Cell Disease, Beta Thalassemia Intermedia

Intervention / Treatment
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* Diagnostic Test: RNA Binding Protein Pumilio1 (PUM1) Expression by reverse transcriptase quantatative PCR


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Patients with sickle cell anemia and β-thalassemia intermedia. Patients are of both sexes (males and females) at any age. Exclusion Criteria: Patients with any other type of haemolytic anaemias. Patients on Hydroxyurea therapy.
Sexes Eligible for Study
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All

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Control group<br>Normal individuals | Diagnostic Test: RNA Binding Protein Pumilio1 (PUM1) Expression by reverse transcriptase quantatative PCR<br>* Quantatative Real -Time PCR for quantification of PUM1; The type of the recruited samples: Ethylenediamine tetra-acetic acid (EDTA) peripheral blood samples. The method of total RNA extraction: TRIzol and TRIzol LS. The purity and concentration of the RNA were measured using a Nano Drop 2000 instrument. cDNA was amplified with primers using the GoScript Reverse Transcription system. Western Blotting Assay of PUM1 protein levels<br>* Other names: Western Blotting Assay of PUM1 protein levels;|
| thalassemia intermedia patients and sickle cell disease patients<br>Inclusion criteria Patients with sickle cell anemia and β-thalassemia intermedia. Patients are of both sexes (males and females) at any age. Exclusion criteria: Patients with any other type of haemolytic anaemias. Patients on Hydroxyurea therapy. | Diagnostic Test: RNA Binding Protein Pumilio1 (PUM1) Expression by reverse transcriptase quantatative PCR<br>* Quantatative Real -Time PCR for quantification of PUM1; The type of the recruited samples: Ethylenediamine tetra-acetic acid (EDTA) peripheral blood samples. The method of total RNA extraction: TRIzol and TRIzol LS. The purity and concentration of the RNA were measured using a Nano Drop 2000 instrument. cDNA was amplified with primers using the GoScript Reverse Transcription system. Western Blotting Assay of PUM1 protein levels<br>* Other names: Western Blotting Assay of PUM1 protein levels;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Expression pattern of PUM1 gene in patients with sickle cell anemia and β-thalassemia intermedia. | Detect PUM1 protein levels in sickle cell anemia and β-thalassemia intermedia patients and to correlate PUM1 gene expression pattern and protein levels with HbF levels in sickle cell anemia and β-thalassemia intermedia patients. | saple taken after diagnosis and before recieve treatment. If patients already on treatment , sample taken at least one month after stoppage of hydroxyurea or blood transfusion |