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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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Abstract

Introduction

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.

Methods

Patient recruitment, genomic DNA sequencing, western blotting, electrophoretic mobility shift assays, surface plasmon resonance (SPR) biospecific interaction analysis, bioinformatics analyses based on docking experiments.

Results

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.

Conclusion

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.

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References

  1. Cao A, Galanello R. Beta-thalassemia. Genet Med. 2010;12:61–76. doi:10.1097/GIM.0b013e3181cd68ed.

    Article  CAS  PubMed  Google Scholar 

  2. Giardine B, Borg J, Viennas E, Pavlidis C, Moradkhani K, Joly P, et al. Updates of the HbVar database of human hemoglobin variants and thalassemia mutations. Nucleic Acids Res. 2014;42 (Database issue):D1063–D1069. doi: 10.1093/nar/gkt911.

  3. Old JM. Screening and genetic diagnosis of haemoglobin disorders. Blood Rev. 2003;17:43–53.

    Article  CAS  PubMed  Google Scholar 

  4. Weatherall DJ. Pathophysiology of thalassaemia. Bailliere’s Clin Haematol. 1998;11:127–46.

    Article  CAS  Google Scholar 

  5. Weatherall DJ. Phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet. 2001;2:245–55.

    Article  CAS  PubMed  Google Scholar 

  6. El-Beshlawy A, Hamdy M, El Ghamrawy M. Fetal globin induction in beta-thalassemia. Hemoglobin. 2009;33:S197–203. doi:10.3109/03630260903351882.

    Article  CAS  PubMed  Google Scholar 

  7. Thein SL. The emerging role of fetal hemoglobin induction in non-transfusion-dependent thalassemia. Blood Rev. 2012;26:S35–9. doi:10.1016/S0268-960X(12)70011-5.

    Article  CAS  PubMed  Google Scholar 

  8. Atweh G, Fathallah HV. Pharmacologic induction of fetal haemoglobin production. Hematol Oncol Clin N. 2010;24:1131–44. doi:10.1016/j.hoc.2010.08.001.

    Article  Google Scholar 

  9. Fibach E, Bianchi N, Borgatti M, Zuccato C, Finotti A, Lampronti I, et al. Effects of rapamycin on accumulation of alpha-, beta- and gamma-globin mRNAs in erythroid precursor cells from beta-thalassaemia patients. Eur J Haematol. 2006;77:437–41.

    Article  CAS  PubMed  Google Scholar 

  10. Fibach E, Prus E, Bianchi N, Zuccato C, Breveglieri G, Salvatori F, et al. Resveratrol: antioxidant activity and induction of fetal hemoglobin in erythroid cells from normal donors and β-thalassemia patients. Int J Mol Med. 2012;29:974–82. doi:10.3892/ijmm.2012.928.

    CAS  PubMed  Google Scholar 

  11. Finotti A, Gambari R. Recent trends for novel options in experimental biological therapy of β-thalassemia. Expert Opin Biol Ther. 2014;14:1443–54. doi:10.1517/14712598.2014.927434.

    Article  CAS  PubMed  Google Scholar 

  12. Gambari R. Alternative options for DNA-based experimental therapy of β-thalassemia. Expert Opin Biol Ther. 2012;12:443–62. doi:10.1517/14712598.2012.665047.

    Article  CAS  PubMed  Google Scholar 

  13. Gambari R, Fibach E. Medicinal chemistry of fetal hemoglobin inducers for treatment of beta-thalassemia. Curr Med Chem. 2007;14:199–212.

    Article  CAS  PubMed  Google Scholar 

  14. Sankaran VG. Targeted therapeutic strategies for fetal hemoglobin induction. Hematol Am Soc Hematol Educ Program. 2011;459–65. doi:10.1182/asheducation-2011.1.459.

  15. Perrine SP, Pace BS, Faller DV. Targeted fetal hemoglobin induction for treatment of beta hemoglobinopathies. Hematol Oncol Clin N. 2014;28:233–48. doi:10.1016/j.hoc.2013.11.009.

    Article  Google Scholar 

  16. Quek L, Thein SL. Molecular therapies in beta-thalassaemia. Br J Haematol. 2007;136:353–65.

    Article  CAS  PubMed  Google Scholar 

  17. Badens C, Joly P, Agouti I, Thuret I, Gonnet K, Fattoum S, et al. Variants in genetic modifiers of β-thalassemia can help to predict the major or intermedia type of the disease. Haematologica. 2011;96:1712–4. doi:10.3324/haematol.2011.046748.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Banan M, Bayat H, Azarkeivan A, Mohammadparast S, Kamali K, Farashi S. The XmnI and BCL11A single nucleotide polymorphisms may help predict hydroxyurea response in Iranian β-thalassemia patients. Hemoglobin. 2012;36:371–80. doi:10.3109/03630269.2012.691147.

    Article  CAS  PubMed  Google Scholar 

  19. Danjou F, Francavilla M, Anni F, Satta S, Demartis FR, Perseu L, et al. A genetic score for the prediction of beta-thalassemia severity. Haematologica. 2015;100:452–7. doi:10.3324/haematol.2014.113886.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Thein SL, Menzel S. Discovering the genetics underlying foetal haemoglobin production in adults. Br J Haematol. 2009;145:455–67. doi:10.1111/j.1365-2141.2009.07650.x.

    Article  CAS  PubMed  Google Scholar 

  21. Jiang J, Best S, Menzel S, Silver N, Lai MI, Surdulescu GL, et al. cMYB is involved in the regulation of fetal hemoglobin production in adults. Blood. 2006;108:1077–83.

    Article  CAS  PubMed  Google Scholar 

  22. Sankaran VG, Menne TF, Xu J, Akie TE, Lettre G, Van Handel B, et al. Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A. Science. 2008;322:1839–42. doi:10.1126/science.1165409.

    Article  CAS  PubMed  Google Scholar 

  23. Thein SL, Menzel S, Lathrop M, Garner C. Control of fetal hemoglobin: new insights emerging from genomics and clinical implications. Hum Mol Genet. 2009;18(R2):R216–23. doi:10.1093/hmg/ddp401.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Trakarnsanga K, Wilson MC, Lau W, Singleton BK, Parsons SF, Sakuntanaga P, et al. Induction of adult levels of β-globin in human erythroid cells that intrinsically express embryonic or fetal globin by transduction with KLF1 and BCL11A-XL. Haematologica. 2014;99:1677–85. doi:10.3324/haematol.2014.110155.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Paciaroni K, Gallucci C, De Angelis G, Alfieri C, Roveda A, Lucarelli G. Sustained and full fetal hemoglobin production after failure of bone marrow transplant in a patient homozygous for beta 0-thalassemia: a clinical remission despite genetic disease and transplant rejection. Am J Hematol. 2009;84:372–3. doi:10.1002/ajh.21392.

    Article  PubMed  Google Scholar 

  26. Paciaroni K, Lucarelli G. Hemopoietic stem cell transplantation failure followed by switch to stable production of fetal hemoglobin. Blood. 2012;119:1091–2. doi:10.1182/blood-2011-10-388678.

    Article  CAS  PubMed  Google Scholar 

  27. Paciaroni K, Lucarelli G, Martelli F, Migliaccio AR, von Lindern M, Borg J, Gillemans N, van Dijk TB, Philipsen S. Transfusion-independent β(0)-thalassemia after bone marrow transplantation failure: proposed involvement of high parental HbF and an epigenetic mechanism. Am J Blood Res. 2014;4:27–32.

    PubMed  PubMed Central  Google Scholar 

  28. Fibach E, Bianchi N, Borgatti M, Prus E, Gambari R. Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells. Blood. 2003;102:1276–81.

    Article  CAS  PubMed  Google Scholar 

  29. Lampronti I, Bianchi N, Zuccato C, Dall’acqua F, Vedaldi D, Viola G, et al. Increase in gamma-globin mRNA content in human erythroid cells treated with angelicin analogs. Int J Hematol. 2009;90:318–27. doi:10.1007/s12185-009-0422-2.

    Article  CAS  PubMed  Google Scholar 

  30. Lozzio CB, Lozzio BB, Machado EA, Fuhr JE, Lair SV, Bamberger EG. Effects of sodium butyrate on human chronic myelogenous leukaemia cell line K562. Nature. 1979;281:709–10.

    Article  CAS  PubMed  Google Scholar 

  31. Bianchi N, Finotti A, Ferracin M, Zuccato C, Breveglieri G, Brognara E, et al. Increase of microRNA-210, decrease of raptor gene expression and alteration of mammalian target of rapamycin regulated proteins following mithramycin treatment of human erythroid cells. PLoS One. 2015;10:1–33. doi:10.1371/journal.pone.0121567.

    Google Scholar 

  32. Finotti A, Treves S, Zorzato F, Gambari R, Feriotto G. Upstream stimulatory factors are involved in the P1 promoter directed transcription of the A β H-J-J locus. BMC Mol Biol. 2008;9:110–25. doi:10.1186/1471-2199-9-110.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991;19:2499. doi:10.1093/nar/19.9.2499.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Borgatti M, Lampronti I, Romanelli A, Pedone C, Saviano M, Bianchi N, et al. Transcription factor decoy molecules based on a peptide nucleic acid (PNA)-DNA chimera mimicking Sp1 binding sites. J Biol Chem. 2002;278:7500–9.

    Article  PubMed  Google Scholar 

  35. Romanelli A, Pedone C, Saviano M, Bianchi N, Borgatti M, Mischiati C, et al. Molecular interactions with nuclear factor kappaB (NF-kappaB) transcription factors of a PNA-DNA chimera mimicking NF-kappaB binding sites. Eur J Biochem. 2001;268:6066–75.

    Article  CAS  PubMed  Google Scholar 

  36. Finotti A, Bianchi N, Fabbri E, Borgatti M, Breveglieri G, Gasparello J, et al. Erythroid induction of K562 cells treated with mithramycin is associated with inhibition of raptor gene transcription and mammalian target of rapamycin complex 1 (mTORC1) functions. Pharmacol Res. 2015;91:57–68. doi:10.1016/j.phrs.2014.11.005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ju J, Wang Y, Liu R, Zhang Y, Xu Z, Wang Y, et al. Human fetal globin gene expression is regulated by LYAR. Nucleic Acids Res. 2014;42:9740–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Stockley PG, Persson B. Surface plasmon resonance assays of DNA-protein interactions. Method Mol Cell Biol. 2009;543:653–69. doi:10.1007/978-1-60327-015-1_38.

    CAS  Google Scholar 

  39. Li ZL, Abe H, Ueki K, Kumagai K, Araki R, Otsuki Y, et al. Identification of c-Jun as bcl-2 transcription factor in human uterine endometrium. J Histochem Cytochem. 2003;51:1601–9.

    Article  CAS  PubMed  Google Scholar 

  40. Marzaro G, Guiotto A, Borgatti M, Finotti A, Gambari R, Breveglieri G, et al. Psoralen derivatives as inhibitors of NF-κB/DNA interaction: synthesis, molecular modeling, 3D-QSAR, and biological evaluation. J Med Chem. 2013;56:1830–42. doi:10.1021/jm3009647.

    Article  CAS  PubMed  Google Scholar 

  41. van Dijk M, Bonvin AM. 3D-DART: a DNA structure modeling server. Nucleic Acids Res. 2009;37(Web Server Issue):W235–W239.

  42. Hess B, Kutzner C, van der Spoel D, Lindahl E. GROMACS 4: algorithms for highly efficient, load-balanced, and scalable molecular simulation. J Chem Theory Comput. 2008;4:435–47. doi:10.1021/ct700301q.

    Article  CAS  PubMed  Google Scholar 

  43. Dominguez C, Boelens R, Bonvin AM. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc. 2003;125:1731–7.

    Article  CAS  PubMed  Google Scholar 

  44. de Vries SJ, van Dijk M, Bonvin AM. The HADDOCK web server for data-driven biomolecular docking. Nat Protocol. 2010;5:883–97. doi:10.1038/nprot.2010.32.

    Article  Google Scholar 

  45. DeLano WL. The PyMOL molecular graphics system. San Carlos: DeLano Scientific; 2002.

    Google Scholar 

  46. Dolinsky TJ, Nielsen JE, McCammon JA, Baker NA. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 2004;32:W665–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Sankaran VG, Xu J, Orkin SH. Transcriptional silencing of fetal hemoglobin by BCL11A. Ann NY Acad Sci. 2010;1202:64–8. doi:10.1111/j.1749-6632.2010.05574.x.

    Article  CAS  PubMed  Google Scholar 

  48. Nguyen TK, Joly P, Bardel C, Moulsma M, Bonello-Palot N, Francina A. The XmnI (G)gamma polymorphism influences hemoglobin F synthesis contrary to BCL11A and HBS1L-MYB SNPs in a cohort of 57 beta-thalassemia intermedia patients. Blood Cel Mol Dis. 2010;45:124–7.

    Article  CAS  Google Scholar 

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Acknowledgments

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.

Author contribution

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.

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Correspondence to Roberto Gambari.

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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.

Funding

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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Bianchi, N., Cosenza, L.C., Lampronti, I. et al. Structural and Functional Insights on an Uncharacterized Aγ-Globin-Gene Polymorphism Present in Four β0-Thalassemia Families with High Fetal Hemoglobin Levels. Mol Diagn Ther 20, 161–173 (2016). https://doi.org/10.1007/s40291-016-0187-2

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