Simultaneous Quantification of Mitochondrial DNA Copy Number and Large-Scale Deletion Using Real-Time PCR Method
Main Article Content
Abstract
Alterations in mitochondrial DNA (mtDNA) copy number and the level of large-scale deletions have been shown to affect the function of the oxidative phosphorylation system (OXPHOS) and are associated with carcinogenesis when they exceed a critical threshold. Most studies quantify either mtDNA copy number alteration or large-scale deletions individually, without assessing both parameters simultaneously. In this study, we aim to develop a real-time PCR-based method capable of simultaneously quantifying mtDNA copy number and the extent of large-scale deletions. The detection limit was established at as low as 20 copies per reaction, with high repeatability. Standard curves demonstrated high linearity (R2 > 0.993) and amplification efficiency (> 93.08%). Application of the method to 20 tissue and blood samples from patients with benign breast tumors revealed that mtDNA copy number was generally higher in tissue samples compared to blood. Conversely, the level of large-scale deletions was significantly lower in tissue than in blood samples (p < 0.05). These findings suggest the successful development of a quantitative method based on real-time PCR with high sensitivity and accuracy, which enables the simultaneous quantification of mtDNA copy number and large-scale deletions, providing a valuable tool for mitochondrial gene analysis in biomedical research.
Keywords:
Large-scale deletion, Mitochondrial DNA, mtDNA copy number, Real-time PCR.
References
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[3] A. Isidoro, M. Martínez, P. L. Fernández, A. D. Ortega, G. Santamaría, M. Chamorro, J. C. Reed, J. M. Cuezva, Alteration of the Bioenergetic Phenotype of Mitochondria is a Hallmark of Breast, Gastric, Lung and Oesophageal Cancer, Biochem J, Vol. 378, No. Pt 1, 2004, pp. 17-20.
[4] Y. Wang, V. W. Liu, W. C. Xue, A. N. Cheung, H. Y. Ngan, Association of Decreased Mitochondrial DNA Content with Ovarian Cancer Progression, Br J Cancer, Vol. 95, No. 8, 2006, pp. 1087-1091.
[5] T. Mizumachi, L. Muskhelishvili, A. Naito, J. Furusawa, C. Y. Fan, E. R. Siegel, F. F. Kadlubar, U. Kumar, M. Higuchi, Increased Distributional Variance of Mitochondrial DNA Content Associated with Prostate Cancer Cells as Compared with Normal Prostate Cells, Prostate, Vol. 68, No. 4, 2008, pp. 408-417.
[6] T. Chen, J. He, L. Shen, H. Fang, H. Nie, T. Jin, X. Wei, Y. Xin, Y. Jiang, H. Li, G. Chen, J. Lu, Y. Bai, The Mitochondrial DNA 4,977-bp Deletion and its Implication in Copy Number Alteration in Colorectal Cancer, BMC Med Genet, Vol. 12, No. 8, 2011.
[7] D. Meierhofer, J. A. Mayr, U. Foetschl, A. Berger, K. Fink, N. Schmeller, G. W. Hacker, C. Hauser-Kronberger, B. Kofler, W. Sperl, Decrease of Mitochondrial DNA Content and Energy Metabolism in Renal Cell Carcinoma, Carcinogenesis, Vol. 25, No. 6, 2004, pp. 1005-1010.
[8] H. C. Lee, S. H. Li, J. C. Lin, C. C. Wu, D. C. Yeh, Y. H. Wei, Somatic Mutations in the D-loop and Decrease in the Copy Number of Mitochondrial DNA in Human Hepatocellular Carcinoma, Mutat Res, Vol. 547, No. 1-2, 2004, pp. 71-78.
[9] C. S. Lin, L. S. Wang, C. M. Tsai, Y. H. Wei, Low Copy Number and Low Oxidative Damage of Mitochondrial DNA are Associated with Tumor Progression in Lung Cancer Tissues after Neoadjuvant Chemotherapy, Interact Cardiovasc Thorac Surg, Vol. 7, No. 6, 2008, pp. 954-958.
[10] S. Compton, C. Kim, N. B. Griner, P. Potluri, I. E. Scheffler, S. Sen, D. J. Jerry, S. Schneider, N. Yadava, Mitochondrial Dysfunction Impairs Tumor Suppressor p53 Expression/Function, J. Biol Chem, Vol. 286, No. 23, 2011, pp. 20297-20312.
[11] D. C. Samuels, E. A. Schon, P. F. Chinnery, Two Direct Repeats Cause most Human MtDNA Deletions, Trends Genet, Vol. 20, No. 9, 2004, pp. 393-398.
[12] G. D. Dakubo, Mitochondrial Genetics and Cancer, Springer Heidelberg Dordrecht London Publishers, New York, 2010.
[13] G. A. Cortopassi, D. Shibata, N. W. Soong, N. Arnheim, A Pattern of Accumulation of a Somatic Deletion of Mitochondrial DNA in Aging Human Tissues, Proc Natl Acad Sci U S A, Vol. 89, No. 16, 1992, pp. 7370-7374.
[14] R. Rossignol, B. Faustin, C. Rocher, M. Malgat, J. P. Mazat, T. Letellier, Mitochondrial Threshold Effects, Biochem J, Vol. 370, No. Pt 3, 2003, pp. 751-762.
[15] R. Filograna, M. Mennuni, D. Alsina, N. G. Larsson, Mitochondrial DNA Copy Number in Human Disease: the More the Better?, FEBS Lett, Vol. 595, No. 8, 2021, pp. 976-1002.
[16] C. A. Castellani, R. J. Longchamps, J. Sun, E. Guallar, D. E. Arking, Thinking Outside the Nucleus: Mitochondrial DNA Copy Number in Health and Disease, Mitochondrion, Vol. 53, 2020, pp. 214 -223.
[17] N. Chen, S. Wen, X. Sun, Q. Fang, L. Huang, S. Liu, W. Li, M. Qiu, Elevated Mitochondrial DNA Copy Number in Peripheral Blood and Tissue Predict the Opposite Outcome of Cancer: A Meta-Analysis, Sci Rep, Vol. 6, No. 37404, 2016.
[18] H. Nie, G. Chen, J. He, F. Zhang, M. Li, Q. Wang, H. Zhou, J. Lyu, Y. Bai, Mitochondrial Common Deletion is Elevated in Blood of Breast Cancer Patients Mediated by Oxidative Stress, Mitochondrion, Vol. 26, 2016, pp. 104-112.
[19] L. He, P. F. Chinnery, S. E. Durham, E. L. Blakely, T. M. Wardell, G. M. Borthwick, R. W. Taylor, D. M. Turnbull, Detection and Quantification of Mitochondrial DNA Deletions in Individual Cells by Real-Time PCR, Nucleic Acids Res, Vol. 30, No. 14, 2002, pp. e68.
[20] A. Lemnrau, M. N. Brook, O. Fletcher, P. Coulson, K. Tomczyk, M. Jones, A. Ashworth, A. Swerdlow, N. Orr, M. Garcia-Closas, Mitochondrial DNA Copy Number in Peripheral Blood Cells and Risk of Developing Breast Cancer, Cancer Res, Vol. 75, No. 14, 2015, pp. 2844-2850.
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[22] P. Xia, H. J. Wang, T. T. Geng, X. J. Xun, W. J. Zhou, T. B. Jin, C. Chen, Mitochondrial DNA Levels in Blood and Tissue Samples from Breast Cancer Patients of Different Stages, Asian Pac J Cancer Prev, Vol. 15, No. 3, 2014, pp. 1339-1344.
[23] R. K. Bai, L. J. Wong, Simultaneous Detection and Quantification of Mitochondrial DNA Deletion(s), Depletion, and Over-Replication in Patients with Mitochondrial Disease, J Mol Diagn, Vol. 7, No. 5, 2005, pp. 613-622.
[24] C. S. Liu, C. S. Tsai, C. L. Kuo, H. W. Chen, C. K. Lii, Y. S. Ma, Y. H. Wei, Oxidative Stress-Related Alteration of the Copy Number of Mitochondrial DNA in Human Leukocytes, Free Radic Res, Vol. 37, No. 12, 2003, pp. 1307-1317.
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[26] D. Meierhofer, J. A. Mayr, S. Ebner, W. Sperl, B. Kofler, Rapid Screening of the Entire Mitochondrial DNA for Low-Level Heteroplasmic Mutations, Mitochondrion, Vol. 5, No. 4, 2005, pp. 282-296.
[27] I. A. Sobenin, K. Y. Mitrofanov, A. V. Zhelankin, M. A. Sazonova, A. Y. Postnov, V. V. Revin, Y. V. Bobryshev, A. N. Orekhov, Quantitative Assessment of Heteroplasmy of Mitochondrial Genome: Perspectives in Diagnostics and Methodological Pitfalls, Biomed Res Int, Vol. 2014, No. 292017, 2014.
[28] C. Ye, X. O. Shu, W. Wen, L. Pierce, R. Courtney, Y. T. Gao, W. Zheng, Q. Cai, Quantitative Analysis of Mitochondrial DNA 4977 bp Deletion in Sporadic Breast Cancer and Benign Breast Diseases, Breast Cancer Res Treat, Vol. 108, No. 3, 2008, pp. 427-434.
[29] E. Mambo, A. Chatterjee, M. Xing, G. Tallini, B. R. Haugen, S. C. Yeung, S. Sukumar, D. Sidransky, Tumor-Specific Changes in MtDNA Content in Human Cancer, Int J Cancer, Vol. 116, No. 6, 2005, pp. 920-924.