Structural and Functional Insights on an Uncharacterized Aγ-Globin-Gene Polymorphism Present in Four β0-Thalassemia Families with High Fetal Hemoglobin Levels
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Several DNA polymorphisms have been associated with high production of fetal hemoglobin (HbF), although the molecular basis is not completely understood. In order to identify and characterize novel HbF-associated elements, we focused on five probands and their four families (from Egypt, Iraq and Iran) with thalassemia major (either β0-IVSII-1 or β0-IVSI-1) and unusual HbF elevation (>98 %), congenital or acquired after rejection of bone marrow transplantation, suggesting an anticipated favorable genetic background to high HbF expression.
Patient recruitment, genomic DNA sequencing, western blotting, electrophoretic mobility shift assays, surface plasmon resonance (SPR) biospecific interaction analysis, bioinformatics analyses based on docking experiments.
A polymorphism of the Aγ-globin gene is here studied in four families with β0-thalassemia (β0-IVSII-1 and β0-IVSI-1) and expressing unusual high HbF levels, congenital or acquired after rejection of bone marrow transplantation. This (G→A) polymorphism is present at position +25 of the Aγ-globin genes, corresponding to a 5′-UTR region of the Aγ-globin mRNA and, when present, is physically linked in chromosomes 11 of all the familiar members studied to the XmnI polymorphism and to the β0-thalassemia mutations. The region corresponding to the +25(G→A) polymorphism of the Aγ-globin gene belongs to a sequence recognized by DNA-binding protein complexes, including LYAR (Ly-1 antibody reactive clone), a zinc-finger transcription factor previously proposed to be involved in down-regulation of the expression of γ-globin genes in erythroid cells.
We found a novel polymorphism of the Aγ-globin gene in four families with β0-thalassemia and high levels of HbF expression. Additionally, we report evidence suggesting that the Aγ-globin gene +25(G→A) polymorphism decreases the efficiency of the interaction between this sequence and specific DNA binding protein complexes.
KeywordsK562 Cell Electrophoretic Mobility Shift Assay Sensor Chip Fetal Hemoglobin Molecular Docking Simulation
The Ferrara Association for the Fight against Thalassemia (ALT) is deeply acknowledged for help in patient recruitment. We thank Dr. Marco Andreani and Manuela Testi (Policlinic of “Tor Vergata” University, Rome, Italy), Dr. Maria Rita Gamberini (Ferrara Hospital, Ferrara, Italy) and Dr. Francesco Chiavilli (Rovigo Hospital, Italy) for providing DNA and cellular samples. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
RG, KP, GL performed the research, designed the research study, wrote the paper and contributed essential reagents or tools; NB performed the research, designed the research study and wrote the paper; LCC, EF, GDA, CG, CA, MR, AI, MM, PS performed the research and analyzed the data; JG and AM designed the research study and analyzed the data; CZ, IL, AF, GB, CG, MB, EF, GM and AC performed the research.
Compliance with Ethical Standards
Conflict of interest
NB, LCC, EF, IL, AF, GB, CZ, GM, AC, GDA, CG, CA, MR, AI, MM, JG, AM, PS,GL, RG and KP declare no conflicts of interest.
Roberto Gambari is funded by Fondazione Cariparo (Cassa di Risparmio di Padova e Rovigo), CIB (Consorzio Interuniversitario per le Biotecnologie), UE THALAMOSS Project (Thalassemia Modular Stratification System for Personalized Therapy of Β-Thalassemia; n. 306201-FP7-HEALTH-2012-INNOVATION-1), Telethon (contract GGP10124) and by COFIN-2010. This research activity has been also supported by Associazione Veneta per la Lotta alla Talassemia (AVLT), Rovigo. Monica Borgatti was funded by Ministero della Salute, Italy (contract 098/GR-2009-1596647). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Ethical approval and informed consent
The present study has been conducted according to the principles expressed in the Declaration of Helsinki and in full compliance with the guidelines of the Mediterranean Institute of Hematology, Rome, Italy. Ethics Committee’s approval for the research has been obtained. All patients and family members provided appropriate informed consent. The study was also approved by the Ethical Committee of Rovigo Hospital and Ferrara Hospital.
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