Abstract:

The purpose of this work was to investigate the expression of iron-regulating genes and their relationship to enzymatic antioxidant genes in thalassaemic patients, as well as to detect distinct forms of thalassaemia using an RT-PCR technique using a presence/absence protocol. This study included 50 patients who were admitted to Mosul's Al-Hadba'a Hospital. Ten healthy subjects and forty thalassaemic patients were aged eight to seventeen years. Freshly blood samples were collected using EDTA for molecular processes, as gene expression and genomic identification of thalassaemia types. The outcomes indicated that the foxO1 gene was significantly upregulated in thalassaemic patients compared to healthy participants, but hepcidin expression was non-significantly downregulated. Similarly, the enzymatic antioxidant gene GSH-Px1 demonstrated significant downstream regulation expression in thalassaemic participants compared to healthy ones, even though the expression of SOD1 and CAT antioxidant enzyme genes did not differ across investigated patients. Catalase expression is associated inversely with foxO1 expression. The presence/absence approach showed that 8.4% and 5.1% of individuals had positive α-thalassaemia based on α1 and α2 mutations in their gDNA samples, respectively, compared to healthy patients. Furthermore, 33.6%, 36.5%, and 16.4% of blood samples with an unknown type of thalassaemia tested positive for ß-thalassaemia because their gDNA samples had codon8/9, codon 41/42, and IVS-I-5 mutations, respectively. Our findings suggest that the RT-PCR approach is the most effective for studying gene expression and molecular identification of thalassaemia types.

References:

[1].   Britton, R. S., Leicester, K. L., Bacon, B. R., 2002, Iron toxicity and chelation therapy. International journal of hematology, 76, 219-228, doi: 10.1007/bf02982791.

[2].   Papanikolaou, G., Tzilianos, M., Christakis, J. I., Bogdanos, D., Tsimirika, K., MacFarlane, J., Nemeth, E., 2005, Hepcidin in iron overload disorders. Blood, 105(10), 4103-4105, doi: 10.1182/BLOOD-2004-12-4844.

[3].   Barisani, D., Pelucchi, S., Mariani, R., Galimberti, S., Trombini, P., Fumagalli, D., Piperno, A., 2008, Hepcidin and iron-related gene expression in subjects with Dysmetabolic Hepatic Iron Overload. Journal of hepatology, 49(1), 123-133, doi: 10.1016/J.JHEP.2008.03.011.

[4].   Latour, C., Kautz, L., Besson‐Fournier, C., Island, M. L., Canonne‐Hergaux, F., Loréal, O., Roth, M. P., 2014, Testosterone perturbs systemic iron balance through activation of epidermal growth factor receptor signaling in the liver and repression of hepcidin. Hepatology, 59(2), 683-694, doi: 10.1002/hep.26648.

[5].   Roth, M. P., Meynard, D., Coppin, H., 2019, Regulators of hepcidin expression. Vitamins and hormones, 110, 101-129, doi: 10.1016/BS.VH.2019.01.005.

[6].   Kassab-Chekir, A., Laradi, S., Ferchichi, S., Khelil, A. H., Feki, M., Amri, F., Miled, A., 2003, Oxidant, antioxidant status and metabolic data in patients with beta-thalassemia. Clinica Chimica Acta, 338(1-2), 79-86, doi: 10.1016/j.cccn.2003.07.010.

[7].   Meral, A., Tuncel, P., Sürmen-Gür, E., Özbek, R., Öztürk, E., GÜnay, Ü., 2000, Lipid peroxidation and antioxidant status in ß-thalassemia. Pediatric hematology and oncology, 17(8), 687-693, doi: 10.1080/08880010050211402.

[8].   Kattamis, C., Lazaropoulou, C., Delaporta, P., Apostolakou, F., Kattamis, A., Papassotiriou, I., 2011, Disturbances of biomarkers of iron and oxidant-antioxidant homeostasis in patients with beta-thalassemia intermedia. Pediatric endocrinology reviews: PER, 8, 256-262.

[9].   Nemeth, E., Ganz, T., 2023, Hepcidin and iron in health and disease. Annual review of medicine, 74(1), 261-277. https://doi.org/10.1146/annurev-med-043021-032816.

[10].  Dolai, T. K., Nataraj, K. S., Sinha, N., Mishra, S., Bhattacharya, M., Ghosh, M. K., 2012, Prevalance of iron deficiency in thalassemia minor: a study from tertiary hospital. Indian Journal of Hematology and Blood Transfusion, 28, 7-9, doi: 10.1007/S12288-011-0088-9/METRICS.

[11].  Rahman, M., Irshadullah, N. M., Ahmed, M., Kabir, A. L., Begum, M., Mostafa, A. G., Khan, A. H., 2014, Prevalence of iron deficiency in thalassemia trait: a study in BSMMU, Dhaka. Bangladesh Journal of Medicine, 25(1), 13-16, doi: 10.3329/BJMED.V25I1.25072.

[12].  Tantiworawit, A., Khemakapasiddhi, S., Rattanathammethee, T., Hantrakool, S., Chai-Adisaksopha, C., Rattarittamrong, E., Fanhchaksai, K., 2021, Correlation of hepcidin and serum ferritin levels in thalassemia patients at Chiang Mai University Hospital. Bioscience Reports, 41(2), BSR20203352, doi: 10.1042/BSR20203352.

[13].  Haghpanah, S., Esmaeilzadeh, M., Honar, N., Hassani, F., Dehbozorgian, J., Rezaei, N., Karimi, M., 2015, Relationship between serum hepcidin and ferritin levels in patients with thalassemia major and intermedia in Southern Iran. Iranian Red Crescent Medical Journal, 17(7), doi: 10.5812/ircmj.17(5)2015.28343.

[14].  Susanah, S., Rakhmilla, L. E., Ghozali, M., Trisaputra, J. O., Moestopo, O., Sribudiani, Y., Maskoen, A. M., 2021, Iron Status in Newly Diagnosed β‐Thalassemia Major: High Rate of Iron Status due to Erythropoiesis Drive. BioMed Research International, 2021(1), 5560319, doi: 10.1155/2021/5560319.

[15].  Eshagh Hossaini, S. K., Haeri, M. R., 2019, Association between serum levels of hepcidin and ferritin in patients with thalassemia major and intermedia, the role of iron chelator. Journal of Hematopathology, 12, 143-147, doi: 10.1007/S12308-019-00363-X/FIGURES/4.

[16].  Lim, W. F., Muniandi, L., George, E., Sathar, J., Teh, L. K., Lai, M. I., 2015, HbF in HbE/β-thalassemia: A clinical and laboratory correlation. Hematology, 20(6), 349-353, doi: 10.1179/1607845414Y.0000000203.

[17].  Voskou, S., Aslan, M., Fanis, P., Phylactides, M., Kleanthous, M., 2015, Oxidative stress in β-thalassaemia and sickle cell disease. Redox biology, 6, 226-239, doi: 10.1016/J.REDOX.2015.07.018.

[18].  Rifkind, J. M., Mohanty, J. G., Nagababu, E., 2015, The pathophysiology of extracellular hemoglobin associated with enhanced oxidative reactions. Frontiers in physiology, 5, 500, doi: 10.3389/FPHYS.2014.00500/BIBTEX.

[19].  Johnson, R. M., Ho, Y. S., Yu, D. Y., Kuypers, F. A., Ravindranath, Y., Goyette, G. W., 2010, The effects of disruption of genes for peroxiredoxin-2, glutathione peroxidase-1, and catalase on erythrocyte oxidative metabolism. Free Radical Biology and Medicine, 48(4), 519-525, doi: 10.1016/J.FREERADBIOMED.2009.11.021.

[20].  Nagababu, E., Mohanty, J. G., Friedman, J. S., Rifkind, J. M., 2013, Role of peroxiredoxin-2 in protecting RBCs from hydrogen peroxide-induced oxidative stress. Free radical research, 47(3), 164-171, doi: 10.3109/10715762.2012.756138.

[21].  Homma, T., Okano, S., Lee, J., Ito, J., Otsuki, N., Kurahashi, T., Fujii, J., 2015, SOD1 deficiency induces the systemic hyperoxidation of peroxiredoxin in the mouse. Biochemical and biophysical research communications, 463(4), 1040-1046, doi: 10.1016/J.BBRC.2015.06.055.

[22].  Al-Allawi, N., Al Allawi, S., Jalal, S. D., 2021, Genetic epidemiology of hemoglobinopathies among Iraqi Kurds. Journal of Community Genetics, 12(1), 5-14, doi: 10.1007/s12687-020-00495-z.

[23].  Suwannakhon, N., Pangeson, T., Seeratanachot, T., Mahingsa, K., Pingyod, A., Bumrungpakdee, W., Sanguansermsri, T., 2019, Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique. Hematology Reports, 11(3), doi: 10.4081/hr.2019.8124.

[24].  Al-Allawi, N. A., Puehringer, H., Raheem, R. A., Oberkanins, C., 2015, Genetic modifiers in β-thalassemia intermedia: a study on 102 Iraqi Arab patients. Genetic testing and molecular biomarkers, 19(5), 242-247, doi: 10.1089/gtmb.2014.0310.

[25].  Kho, S. L., Chua, K. H., George, E., Tan, J.A.M. A., 2015, A novel gap-PCR with high resolution melting analysis for the detection of α-thalassaemia Southeast Asian and Filipino β0-thalassaemia deletion. Scientific Reports, 5, doi: 10.1038/srep13937.

[26].  Lazarte, S. S., Mónaco, M. E., Haro, A. C., Jiménez, C. L., Ledesma Achem, M. E., Issé, B. A., 2014, Molecular characterization and phenotypical study of β-thalassemia in Tucumán, Argentina. Hemoglobin, 38(6), 394-401, doi: 10.3109/03630269.2014.968784.

[27].  Saud, A. M., 2012, Molecular and biochemical study on β-thalassemia patients in Iraq. University of Baghdad.pdf

[28].  Marashi, S. J., Eshkoor, S. A., saed Mirinargesi, M., Sarookhani, M. R., Rahmat, A. B., Ismail, P. B., 2012, Detection of eight common [beta]-globin gene mutation in thalassemia major patients using real time polymerase chain reaction (PCR)-high resolution melting and EvaGreen (TM) dye. African Journal of Biotechnology, 11(2), 448, doi: 10.5897/ajb10.1167.

[29].  Al-Allawi, N. A., Badi, A. I., Imanian, H., Nikzat, N., Jubrael, J. M., Najmabadi, H., 2009, Molecular characterization of α-thalassemia in the Dohuk region of Iraq. Hemoglobin, 33(1), 37-44, doi: 10.1080/03630260802626053.

[30].  Pornprasert, S., Phusua, A., Suanta, S., Saetung, R., Sanguansermsri, T., 2008, Detection of alpha‐thalassemia‐1 Southeast Asian type using real‐time gap‐PCR with SYBR Green1 and high resolution melting analysis. European journal of haematology, 80(6), 510-514, doi: 10.1111/j.1600-0609.2008.01055.x.

[31].  Gaafar, T. M., ELBeshlawy, A. M., Aziz, M. I., Abdelrazik, H. N., 2006, Rapid screening of β-Globin gene mutations by Real-Time PCR in Egyptian thalassemic children. African Journal of Health Sciences, 13(3), 70-77, doi: 10.4314/ajhs.v13i3.30839.

[32].  Al-Allawi, N. A., Jubrael, J. M., Hughson, M., 2006, Molecular characterization of β-thalassemia in the Dohuk region of Iraq. Hemoglobin, 30(4), 479-486, doi: 10.1080/03630260600868097.

[33].  Sun, C. F., Lee, C. H., Cheng, S. W., Lin, M. H., Wu, T. L., Tsao, K. C., Chu, D. C., 2001, Real‐time quantitative PCR analysis for α‐thalassemia‐1 of Southeast Asian type deletion in Taiwan. Clinical genetics, 60(4), 305-309, doi: 10.1034/j.1399-0004.2001.600409.x.

[34].  Oron‐Karni, V., Filon, D., Oppenheim, A., Rund, D., 1998, Rapid detection of the common Mediterranean α‐globin deletions/rearrangements using PCR. American journal of hematology, 58(4), 306-310, doi: 10.1002/(SICI)1096-8652(199808)58:4<306::AID-AJH10>3.0.CO;2-5.

[35].  Chang, J. G., Liu, H. J., Huang, J. M., Yang, T. Y., Chang, C. P., 1997, Multiplex mutagenically separated PCR: diagnosis of β-thalassemia and hemoglobin variants. Biotechniques, 22(3), 520-527, doi: 10.2144/97223rr03.

[36].  Rosnah, B., Rosline, H., Zaidah, A. W., Noor Haslina, M. N., Marini, R., Shafini, M. Y., Nurul Ain, F. A., 2012, Detection of Common Deletional Alpha‐Thalassemia Spectrum by Molecular Technique in Kelantan, Northeastern Malaysia. International Scholarly Research Notices, 2012(1), 462969, doi: 10.5402/2012/462969.

[37].  Farra, C., Badra, R., Fares, F., Muwakkit, S., Dbaibo, G., Dabbous, I., Abboud, M. R., 2015, Alpha thalassemia allelic frequency in Lebanon. Pediatric Blood & Cancer, 62(1), 120-122, doi: 10.1002/PBC.25242.

[38].  Hadavi, V., Taromchi, A. H., Malekpour, M., Gholami, B., Law, H. Y., Almadani, N., Najmabadi, H., 2007, Elucidating the spectrum of α-thalassemia mutations in Iran. haematologica, 92(7), 992-993, doi: 10.3324/HAEMATOL.10658.

[39].  Adekile, A., Sukumaran, J., Thomas, D., D’Souza, T., Haider, M., 2020, Alpha thalassemia genotypes in Kuwait. BMC Medical Genetics, 21, 1-5, doi: 10.1186/S12881-020-01105-Y/TABLES/4.

[40].  Baysal, E., 2011, α-Thalassemia syndromes in the United Arab Emirates. Hemoglobin, 35(5-6), 574-580, doi: 10.3109/03630269.2011.634698.