Abstract
Mutations in mitochondrial DNA (mtDNA) may result in various pathological processes. Detection of mutant mtDNAs is a problem for diagnostic practice that is complicated by heteroplasmy – a phenomenon of the inferring presence of at least two allelic variants of the mitochondrial genome. Also, the level of heteroplasmy largely determines the profile and severity of clinical manifestations. Here we discuss detection of mutations in heteroplasmic mtDNA using up-todate methods that have not yet been introduced as routine clinical assays. These methods can be used for detecting mutations in mtDNA to verify diagnosis of “mitochondrial disease”, studying dynamics of mutant mtDNA in body tissues of patients, as well as investigating structural features of mtDNAs. Original data on allele-specific discrimination of m.11778G>A mutation by droplet digital PCR are presented, which demonstrate an opportunity for simultaneous detection and quantitative assessment of mutations in mtDNAs.
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Chinnery, P. F., Howell, N., Andrews, R. M., and Turnbull, D. M. (1999) Clinical mitochondrial genetics, J. Med. Genet., 36, 425–436.
Anderson, S., Bankier, A. T., Barrell, B. G., De Bruijn, M. H., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J., Staden, R., and Young, I. G. (1981) Sequence and organization of the human mitochondrial genome, Nature, 290, 457–465.
Chinnery, P. F., and Schon, E. A. (2003) Mitochondria, J. Neurol. Neurosurg. Psychiatry, 74, 1188–1199.
Wachsmuth, M., Hubner, A., Li, M., Madea, B., and Stoneking, M. (2016) Age-related and heteroplasmy-related variation in human mtDNA copy number, PLoS Genet., 12, e1005939.
Gorman, G. S., Schaefer, A. M., Ng, Y., Gomez, N., Blakely, E. L., Alston, C. L., Feeney, C., Horvath, R., YuWai-Man, P., Chinnery, P. F., Taylor, R. W., Turnbull, D. M., and McFarland, R. (2015) Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease, Ann. Neurol., 77, 753–759.
Pfeffer, G., and Chinnery, P. F. (2013) Diagnosis and treatment of mitochondrial myopathies, Ann. Med., 45, 4–16.
Pfeffer, G., Horvath, R., Klopstock, T., Mootha, V. K., Suomalainen, A., Koene, S., Hirano, M., Zeviani, M., Bindoff, L. A., Yu-Wai-Man, P., Hanna, M., Carelli, V., McFarland, R., Majamaa, K., Turnbull, D. M., Smeitink, J., and Chinnery, P. F. (2013) New treatments for mitochondrial disease-no time to drop our standards, Nat. Rev. Neurol., 9, 474–481.
Hyslop, L. A., Blakeley, P., Craven, L., Richardson, J., Fogarty, N. M., Fragouli, E., Lamb, M., Wamaitha, S. E., Prathalingam, N., Zhang, Q., O’Keefe, H., Takeda, Y., Arizzi, L., Alfarawati, S., Tuppen, H. A., Irving, L., Kalleas, D., Choudhary, M., Wells, D., Murdoch, P., Turnbull, D. M., Niakan, K. K., and Herbert, M. (2016) Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease, Nature, doi: 10.1038/nature18303.
Bredenoord, A. L., Dondorp, W., Pennings, G., and De Wert, G. (2010) Avoiding transgenerational risks of mitochondrial DNA disorders: a morally acceptable reason for sex selection? Hum. Reprod., 25, 1354–1360.
Ylikallio, E., and Suomalainen, A. (2012) Mechanisms of mitochondrial diseases, Ann. Med., 44, 41–59.
Bacman, S. R., Williams, S. L., Pinto, M., Peralta, S., and Moraes, C. T. (2013) Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs, Nat. Med., 19, 1111–1113.
Gammage, P. A., Rorbach, J., Vincent, A. I., Rebar, E. J., and Minczuk, M. (2014) Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations, EMBO Mol. Med., 6, 458–466.
Jo, A., Ham, S., Lee, G. H., Lee, Y. I., Kim, S., Lee, Y. S., Shin, J. H., and Lee, Y. (2015) Efficient mitochondrial genome editing by CRISPR/Cas9, Biomed Res. Int., doi: 10.1155/2015/305716.
Orishchenko, K. E., Sofronova, Yu. K., Chupakhin, E. G., Lunev, E. A., and Mazunin, I. O. (2016) Delivery of Cas9 into mitochondria, Genes Cells, 11, in press.
Chinnery, P. F. (2016) Mitochondrial disease in adults: what’s old and what’s new? EMBO Mol. Med., 12, 1503–1512.
Koopman, W. J., Beyrath, J., Fung, C. W., Koene, S., Rodenburg, R. J., Willems, P. H., and Smeitink, J. A. (2016) Mitochondrial disorders in children: toward development of small-molecule treatment strategies, EMBO Mol. Med., 8, 311–327.
Suomalainen, A., Elo, J. M., Pietilainen, K. H., Hakonen, A. H., Sevastianova, K., Korpela, M., Isohanni, P., Marjavaara, S. K., Tyni, T., Kiuru-Enari, S., Pihko, H., Darin, N., Ounap, K., Kluijtmans, L. A., Paetau, A., Buzkova, J., Bindoff, L. A., Annunen-Rasila, J., Uusimaa, J., Rissanen, A., Yki-Jarvinen, H., Hirano, M., Tulinius, M., Smeitink, J., and Tyynismaa, H. (2011) FGF-21 as a biomarker for muscle-manifesting mitochondrial respiratory chain deficiencies: a diagnostic study, Lancet Neurol., 9, 806–818.
Ji, K., Zheng, J., Lv, J., Xu, J., Ji, X., Luo, Y. B., Li, W., Zhao, Y., and Yan, C. (2015) Skeletal muscle increases FGF21 expression in mitochondrial disorders to compensate for energy metabolic insufficiency by activating the mTOR-YY1-PGC1α pathway, Free Radic. Biol. Med., 84, 161–170.
Wong, L. J., Scaglia, F., Graham, B. H., and Craigen, W. J. (2010) Current molecular diagnostic algorithm for mitochondrial disorders, Mol. Genet. Metab., 100, 111–117.
Ma, Y., Fang, F., Yang, Y., Zou, L., Zhang, Y., Wang, S., Xu, Y., Pei, P., and Qi, Y. (2009) The study of mitochondrial A3243G mutation in different samples, Mitochondrion, 9, 139–143.
Kozlowski, P., Jasinska, A. J., and Kwiatkowski, D. J. (2008) New applications and developments in the use of multiplex ligation-dependent probe amplification, Electrophoresis, 23, 4627–4636.
Mayorga, L., Laurito, S. R., Loos, M. A., Eiroa, H. D., De Pinho, S., Lubieniecki, F., Arroyo, H. A., Pereyra, M. F., Kauffman, M. A., and Roque, M. (2016) Mitochondrial DNA deletions detected by multiplex ligation-dependent probe amplification, Mitochondrial DNA A DNA MappSeq. Anal., 27, 2864–2867
Vasta, V., Ng, S. B., Turner, E. H., Shendure, J., and Hahn, S. H. (2009) Next generation sequence analysis for mitochondrial disorders, Genome Med., 23, 100.
Palculict, M. E., Zhang, V. W., Wong, L. J., and Wang, J. (2016) Comprehensive mitochondrial genome analysis by massively parallel sequencing, Methods Mol. Biol., 1351, 3–17.
Moraes, C. T., Atencio, D. P., Oca-Cossio, J., and Diaz, F. (2003) Techniques and pitfalls in the detection of pathogenic mitochondrial DNA mutations, J. Mol. Diagn., 5, 197–208.
Kurelac, I., Lang, M., Zuntini, R., Calabrese, C., Simone, D., Vicario, S., Santamaria, M., Attimonelli, M., Romeo, G., and Gasparre, G. (2012) Searching for a needle in the haystack: comparing six methods to evaluate heteroplasmy in difficult sequence context, Biotechnol. Adv., 30, 363–371.
Sobenin, I. A., Mitrofanov, K. Y., Zhelankin, A. V., Sazonova, M. A., Postnov, A. Y., Revin, V. V., Bobryshev, Y. V., and Orekhov, A. N. (2014) Quantitative assessment of heteroplasmy of mitochondrial genome: perspectives in diagnostics and methodological pitfalls, Biomed Res. Int., 292017.
Hindson, B. J., Ness, K. D., Masquelier, D. A., Belgrader, P., Heredia, N. J., Makarewicz, A. J., Bright, I. J., Lucero, M. Y., Hiddessen, A. L., Legler, T. C., Kitano, T. K., Hodel, M. R., Petersen, J. F., Wyatt, P. W., Steenblock, E. R., Shah, P. H., Bousse, L. J., Troup, C. B., Mellen, J. C., Wittmann, D. K., Erndt, N. G., Cauley, T. H., Koehler, R. T., So, A. P., Dube, S., Rose, K. A., Montesclaros, L., Wang, S., Stumbo, D. P., Hodges, S. P., Romine, S., Milanovich, F. P., White, H. E., Regan, J. F., KarlinNeumann, G. A., Hindson, C. M., Saxonov, S., and Colston, B. W. (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number, Anal. Chem., 83, 8604–8610.
Podlesniy, P., Figueiro-Silva, J., Llado, A., Antonell, A., Sanchez-Valle, R., Alcolea, D., Lleo, A., Molinuevo, J. L., Serra, N., and Trullas, R. (2013) Low cerebrospinal fluid concentration of mitochondrial DNA in preclinical Alzheimer’s disease, Ann. Neurol., 74, 655–668.
Wachsmuth, M., Hubner, A., Li, M., Madea, B., and Stoneking, M. (2016) Age-related and heteroplasmy-related variation in human mtDNA copy number, PLoS Genet., 12, e1005939.
Taylor, S. D., Ericson, N. G., Burton, J. N., Prolla, T. A., Silber, J. R., Shendure, J., and Bielas, J. H. (2014) Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain, Aging Cell, 13, 29–38.
Rebolledo-Jaramillo, B., Su, M. S., Stoler, N., McElhoe, J. A., Dickins, B., Blankenberg, D., Korneliussen, T. S., Chiaromonte, F., Nielsen, R., Holland, M. M., Paul, I. M., Nekrutenko, A., and Makova, K. D. (2014) Maternal age effect and severe germ-line bottleneck in the inheritance of human mitochondrial DNA, Proc. Natl. Acad. Sci. USA, 111, 15474–15479.
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Original Russian Text © J. K. Sofronova, Y. Y. Ilinsky, K. E. Orishchenko, E. G. Chupakhin, E. A. Lunev, I. O. Mazunin, 2016, published in Biokhimiya, 2016, Vol. 81, No. 10, pp. 1293–1298.
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Sofronova, J.K., Ilinsky, Y.Y., Orishchenko, K.E. et al. Detection of mutations in mitochondrial DNA by droplet digital PCR. Biochemistry Moscow 81, 1031–1037 (2016). https://doi.org/10.1134/S0006297916100011
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DOI: https://doi.org/10.1134/S0006297916100011