Advertisement

Biochemistry (Moscow)

, Volume 81, Issue 10, pp 1031–1037 | Cite as

Detection of mutations in mitochondrial DNA by droplet digital PCR

  • J. K. Sofronova
  • Y. Y. Ilinsky
  • K. E. Orishchenko
  • E. G. Chupakhin
  • E. A. Lunev
  • I. O. MazuninEmail author
Molecular Biology of Mitochondria (Special Issue) Guest Editor — P. A. Kamenski

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.

Key words

mitochondria mutations mitochondrial diseases heteroplasmy droplet digital PCR 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Chinnery, P. F., Howell, N., Andrews, R. M., and Turnbull, D. M. (1999) Clinical mitochondrial genetics, J. Med. Genet., 36, 425–436.PubMedPubMedCentralGoogle Scholar
  2. 2.
    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.CrossRefPubMedGoogle Scholar
  3. 3.
    Chinnery, P. F., and Schon, E. A. (2003) Mitochondria, J. Neurol. Neurosurg. Psychiatry, 74, 1188–1199.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Pfeffer, G., and Chinnery, P. F. (2013) Diagnosis and treatment of mitochondrial myopathies, Ann. Med., 45, 4–16.CrossRefPubMedGoogle Scholar
  7. 7.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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.Google Scholar
  9. 9.
    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.CrossRefPubMedGoogle Scholar
  10. 10.
    Ylikallio, E., and Suomalainen, A. (2012) Mechanisms of mitochondrial diseases, Ann. Med., 44, 41–59.CrossRefPubMedGoogle Scholar
  11. 11.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    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.Google Scholar
  14. 14.
    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.Google Scholar
  15. 15.
    Chinnery, P. F. (2016) Mitochondrial disease in adults: what’s old and what’s new? EMBO Mol. Med., 12, 1503–1512.Google Scholar
  16. 16.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    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.CrossRefGoogle Scholar
  18. 18.
    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.CrossRefPubMedGoogle Scholar
  19. 19.
    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.CrossRefPubMedGoogle Scholar
  20. 20.
    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.CrossRefPubMedGoogle Scholar
  21. 21.
    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.CrossRefGoogle Scholar
  22. 22.
    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, 2864–2867PubMedGoogle Scholar
  23. 23.
    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.CrossRefGoogle Scholar
  24. 24.
    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.CrossRefPubMedGoogle Scholar
  25. 25.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    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.CrossRefPubMedGoogle Scholar
  27. 27.
    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.Google Scholar
  28. 28.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    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.CrossRefPubMedGoogle Scholar
  30. 30.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    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.CrossRefPubMedGoogle Scholar
  32. 32.
    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.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2016

Authors and Affiliations

  • J. K. Sofronova
    • 1
  • Y. Y. Ilinsky
    • 1
    • 2
    • 3
  • K. E. Orishchenko
    • 1
    • 2
  • E. G. Chupakhin
    • 1
  • E. A. Lunev
    • 1
  • I. O. Mazunin
    • 1
    Email author
  1. 1.Immanuil Kant Baltic Federal UniversityInstitute of Chemistry and BiologyKaliningradRussia
  2. 2.Federal Research Center Institute of Cytology and GeneticsSiberian Branch of the Russian Academy of SciencesNovosibirskRussia
  3. 3.Novosibirsk State UniversityNovosibirskRussia

Personalised recommendations