Abstract

Hemoglobinopathies, characterized by abnormal hemoglobin structure or production, are primarily caused by mutations in the HBB gene located on chromosome 11. While the contribution of nuclear gene mutations is welldocumented, the involvement of mitochondrial DNA (mtDNA) variations, particularly within the D-loop region, remains underexplored in antenatal carriers. This study screened 320 blood samples from pregnant women to identify the antenatal carriers for hemoglobinopathy by assessing abnormal hemoglobin levels using hemoglobin electrophoresis, and investigated the association between HBB mutations and mitochondrial D-loop variations in 18 hemoglobinopathy carriers using Sanger sequencing. This analysis identified IVS1-5G>C as the most prevalent HBB mutation (83.3%), followed by c.6 A>T and c.26 G>A. Four novel heteroplasmic variations in the Mitochondrial D-loop were identified at positions 716A>C, 747A>T, 749G>C, and 786G>C. The variation at position 786 was associated with the c26 mutations (HbE variant), while the others were linked to the IVS1-5 mutation (Beta thalassemia trait, HbA2). These novel D-loop variations, absent in current GenBank records, may serve as potential genetic markers influencing disease susceptibility and clinical phenotype in antenatal hemoglobinopathy carriers. Our findings indicate that mitochondrial D-loop variations could play a pivotal role in the genetic architecture of hemoglobinopathy in antenatal carriers, potentially influencing disease expression and providing insights into genetic counseling. Further research is needed to clarify the interplay between nuclear and mitochondrial genomes and their implications for antenatal screening and management strategies in hemoglobinopathy and related genetic disorders.

• 1.   Ansharullah, B.A., Sutanto, H., Romadhon, P.Z., 2025. Thalassemia and iron overload cardiomyopathy: Pathophysiological insights, clinical implications, and management strategies. Curr Probl Cardiol 50, 102911. https:// doi.org/10.1016/J.CPCARDIOL.2024.102911.
• 2.   Aramayo-Singelmann, C., Halimeh, S., Proske, P., Vignalingarajah, A., Cario, H., Christensen, M.O., Yamamoto, R., Röth, A., Reinhardt, D., Reinhardt, H.C., Alashkar, F., 2022. Screening and diagnosis of hemoglobinopathies in Germany: Current state and future perspectives. Sci Rep 12. https://doi.org/10.1038/S41598- 022-13751-8.
• 3.   Athiya Munuvarah, S., 2010. Nestroft as a Screening Tool to Identify Beta Thalassaemia Trait in Paediatric Cases Dissertation Submitted.
• 4.   Dehury, S., Purohit, P., Meher, S., Das, K., Patel, S., 2015. Compound heterozygous state of β-thalassemia with IVS1-5 (G→C) mutation and Indian deletion-inversion Gγ(Aγδβ)0 - thalassemia in eastern India. Rev Bras Hematol Hemoter 37, 202–206. https://doi. org/10.1016/J.BJHH.2014.12.002.
• 5.   Dordevic, A., Mrakovcic-Sutic, I., Pavlovic, S., Ugrin, M., Roganovic, J., 2025. Beta thalassemia syndromes: New insights. World J Clin Cases 13, 100223. https://doi.org/10.12998/WJCC. V13.I10.100223.
• 6.   Ferro, E., Visalli, G., Civa, R., La Rosa, M.A., Randazzo Papa, G., Baluce, B., D’Ascola, D.G., Piraino, B., Salpietro, C., Di Pietro, A., 2012. Oxidative damage and genotoxicity biomarkers in transfused and untransfused thalassemic subjects. Free Radic Biol Med 53, 1829–1837. https://doi.org/10.1016/J. FREERADBIOMED.2012.08.592.
• 7.   Gardenghi, S., Grady, R.W., Rivella, S., 2010. Anemia, ineffective erythropoiesis, and hepcidin: Interacting factors in abnormal iron metabolism leading to iron overload in β-thalassemia. Hematol Oncol Clin North Am. https://doi.org/10.1016/j.hoc.2010.08.003.
• 8.   Guo, X.G., Guo, Q.N., 2006. Mutations in the mitochondrial DNA D-Loop region occur frequently in human osteosarcoma. Cancer Lett 239, 151–155. https://doi.org/10.1016/J. Canlet.2005.08.008.
• 9.   Iyer, S., Sakhare, S., Sengupta, C., Velumani, A., 2015. Hemoglobinopathy in India. Clin Chim Acta 444, 229–233. https://doi.org/10.1016/J. CCA.2015.02.033.
• 10.   Khan, M.B.N., Iftikhar, F., Khan, T., Danish, A., Shamsi, T., Musharraf, S.G., Siddiqui, A.J., 2022. IVS I-5 (G > C) is associated with changes to the RBC membrane lipidome in response to hydroxyurea treatment in β-thalassemia patients. Mol Omics 18, 534–544. https://doi. org/10.1039/D2MO00008C.
• 11.   Langer, A.L., 2024. Beta-Thalassemia. GeneReviews®.
• 12.   Laosombat, V., Wongchanchailert, M., Sattayasevana, B., Wiriyasateinkul, A., Fucharoen, S., 2001. Clinical and hematological features of beta(+)-thalassemia (IVS1 nt 5, G-C mutation) in Thai patients. Eur J Haematol 67, 100–104. https://doi. org/10.1034/J.1600-0609.2001.T01-1-00431.X.
• 13.   Lo, L., Singer, S.T., 2002. Thalassemia: current approach to an old disease. Pediatr Clin North Am 49, 1165–1191. https://doi.org/10.1016/ S0031-3955(02)00088-3
• 14.   Nadalutti, C.A., Ayala-Peña, S., Santos, J.H., 2022. Mitochondrial DNA damage as driver of cellular outcomes. Am J Physiol Cell Physiol 322, C136–C150. https://doi.org/10.1152/ AJPCELL.00389.2021/Asset/Images/Large/ AJPCELL.00389.2021_F004.JPEG.
• 15.   Narahari, J.M., Guruswamy, P., Jagadeesha, N.M., Shivashakar, K.K., Kumar, D.P., Narayana, P.S., Vishwanath, P.M., Prashant, A., 2024. Exploring the Impact of Iron Overload on Mitochondrial DNA in β-Thalassemia: A Comprehensive Review. Gene Expression The Journal of Liver Research 23, 197–210. https:// doi.org/10.14218/GE.2023.00128.
• 16.   Nie, G., Sheftel, A.D., Kim, S.F., Ponka, P., 2005. Overexpression of mitochondrial ferritin causes cytosolic iron depletion and changes cellular iron homeostasis. Blood 105, 2161–2167. https://doi.org/10.1182/Blood-2004-07-2722.
• 17.   Satoh, M., Kuroiwa, T., 1991. Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell. Exp Cell Res 196, 137–140. https://doi.org/10.1016/0014- 4827(91)90467-9.
• 18.   Sharma, H., Singh, A., Sharma, C., Jain, S.K., Singh, N., 2005. Mutations in the mitochondrial DNA D-loop region are frequent in cervical cancer. Cancer Cell Int 5, 34. https://doi. org/10.1186/1475-2867-5-34.
• 19.   Singh, H., Kumar, S., Urs, A.B., Kapoor, S., 2022. Identification of sequence polymorphisms in the D-loop region of mitochondrial DNA as valuable biomarkers for salivary gland tumors: an observational study. Egyptian Journal of Otolaryngology 38. https://doi.org/10.1186/ S43163-022-00208-Y.
• 20.   Singh, V., Biswas, A.K., Baranwal, A.K., Asthana, B., Dahiya, T., 2024. Prevalence of hemoglobinopathies using highperformance liquid chromatography as diagnostic tool in anemic patients of tertiary care center of Western India. Asian J Transfus Sci 18, 257–263. https://doi.org/10.4103/AJTS.AJTS_62_22.
• 21.   Somervaille, T., 2001. Disorders of Hemoglobin: Genetics, Pathophysiology, and Clinical Management. J R Soc Med 94, 602.
• 22.   Suhail, M., 2024. Biophysical chemistry behind sickle cell anemia and the mechanism of voxelotor action. Sci Rep 14, 1861. https://doi. org/10.1038/S41598-024-52476-8.
• 23.   Takamatsu, C., Umeda, S., Ohsato, T., Ohno, T., Abe, Y., Fukuoh, A., Shinagawa, H., Hamasaki, N., Kang, D., 2002. Regulation of mitochondrial D-loops by transcription factor A and single-stranded DNA-binding protein. Embo Rep 3, 451. https://doi.org/10.1093/Embo-Reports/ KVF099.
• 24.   Thein, S.L., 2018. Molecular basis of β thalassemia and potential therapeutic targets. Blood Cells Mol Dis. https://doi. org/10.1016/j.bcmd.2017.06.001.
• 25.   Tyagi, A., Pramanik, R., Vishnubhatla, S., Bakhshi, R., Bakhshi, S., 2019. Prognostic impact of mitochondrial DNA D-loop variations in pediatric acute myeloid leukemia. Oncotarget 10, 1334–1343. https://doi.org/10.18632/ Oncotarget.26665.
• 26.   Williams, T.N., Weatherall, D.J., 2012. World Distribution, Population Genetics, and Health Burden of the Hemoglobinopathies. Cold Spring Harb Perspect Med 2, a011692. https:// doi.org/10.1101/Cshperspect.A011692.
• 27.   Zhao, Y., Yang, M., Liang, X., 2024. The role of mitochondria in iron overload-induced damage. J Transl Med 22, 1057. https://doi. org/10.1186/S12967-024-05740-4

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