Analysis of Mitochondrial DNA Mutations

  • Robert W. Taylor
  • Theresa M. Wardell
  • Emma L. Blakely
  • Gillian M. Borthwick
  • Elizabeth J. Brierley
  • Douglass M. Turnbull
Part of the Methods in Molecular Medicine book series (MIMM, volume 38)


Although the precise mechanisms of the aging process remain poorly understood, a plausible theory for cellular dysfunction and deterioration during aging involves mitochondria (1, 2). The major function of mitochondria is to generate energy for cellular processes in the form of ATP by oxidative phosphorylation. Mitochondria contain their own DNA (mtDNA), a small 16.5 kb circular molecule that encodes 13 essential polypeptides of the mitochondrial respiratory chain, as well as 2 rRNAs and 22 tRNAs required for intramitochondrial protein synthesis (3). The mitochondrial respiratory chain is a series of five, multisubunit protein complexes located within the inner mitochondrial membrane. The first four of these (complexes I-IV) reoxidize reduced cofactors (NADH and FADH2) generated by the oxidation of foodstuffs, thereby generating an electrochemical gradient across the inner mitochondrial membrane which is harnessed by the fifth complex, the ATP synthetase, to drive the formation of ATP.


Polymerase Chain Reaction Polymerase Chain Reaction Product Polymerase Chain Reaction Amplification Polymerase Chain Reaction Reaction Inclusion Body Myositis 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Harman, D. (1972) The biologic clock: the mitochondria? J. Am. Geriatr. Soc. 20, 145–147.PubMedGoogle Scholar
  2. 2.
    Fleming, J. E., Miquel, J., Cottrell, S. F., Yengoyan, L. S., and Economos, A. S. (1982) Is cell aging caused by respiration-dependent injury to the mitochondrial genome? Gerontology 28, 44–53.PubMedCrossRefGoogle Scholar
  3. 3.
    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 organisation of the human mitochondrial genome. Nature 290, 457–465.PubMedCrossRefGoogle Scholar
  4. 4.
    Cortopassi, G. A. and Arnheim, N. (1990) Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucleic Acids Res. 18, 6927–6933.PubMedCrossRefGoogle Scholar
  5. 5.
    Ozawa, T., Tanaka, M., Ikebe, S., Ohno, K., Kondo, T., and Mizuno, Y. (1990) Quantitative determination of deleted mitochondrial DNA relative to normal DNA in parkinsonian striatum by a kinetic PCR analysis. Biochem. Biophys Res. Commun. 172, 483–489.PubMedCrossRefGoogle Scholar
  6. 6.
    Hattori, K., Tanaka, M., Sugiyama, S., Obayashi, T., Ito, T., Satake, T., Hanaki, Y., Assai, J., Nagano, M., and Ozawa, T. (1991) Age-dependent increase in deleted mitochondrial DNA in the human heart: possible contribution factor in presbycardia. Am. Heart J. 121, 1735–1742.PubMedCrossRefGoogle Scholar
  7. 7.
    Corral-Debrinski, M., Stepien, G., Shoffner, J. M., Lott, M. T., Kanter, K., and Wallace, D. C. (1991) Hypoxemia is associated with mitochondrial DNA damage and gene induction: implications for cardiac disease. JAMA 266, 1812–1816.PubMedCrossRefGoogle Scholar
  8. 8.
    Corral-Debrinski, M., Shoffner, J. M., Lott, M. T., and Wallace, D. C. (1992) Association of mitochondrial DNA damage with aging and coronary atherosclerotic heart disease. Mutat. Res. 275, 169–180.PubMedGoogle Scholar
  9. 9.
    Corral-Debrinski, M., Horton, T., Lott., M. T., Shoffner, J. M., Beal, M. F., and Wallace, D. C. (1992) Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age. Nature Genet. 2, 324–329.PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang, C., Baumer, A., Maxwell, R. J., Linnane, A. W., and Nagley, P. (1992) Multiple mitochondrial DNA deletions in an elderly human individual. FEBS Lett. 297, 34–38.PubMedCrossRefGoogle Scholar
  11. 11.
    Simonetti, S., Chen, X., DiMauro, S., and Schon, E. A. (1992) Accumulation of deletions in human mitochondrial DNA during normal aging: analysis by quantitative PCR. Biochim. Biophys. Acta 1180, 113–122.PubMedGoogle Scholar
  12. 12.
    Cooper, J. M., Mann, V. M., and Schapira, A. H. (1992) Analyses of mitochondrial respiratory chain function and mitochondrial DNA deletion in human skeletal muscle: effect of ageing. J. Neurol. Sci. 113, 91–98.PubMedCrossRefGoogle Scholar
  13. 13.
    Baumer, A., Zhang, C., Linnane, A. W., and Nagley, P. (1994) Age-related human mtDNA deletions: a heterogeneous set of deletions arising at a single pair of directly repeated sequences. Am. J. Hum. Genet. 54, 618–630.PubMedGoogle Scholar
  14. 14.
    Lee, H. C., Pang, C. Y., Hsu, H. S., and Wei, Y. H. (1994) Differential accumulations of 4, 977 bp deletion in mitochondrial DNA of various tissues in human ageing. Biochim. Biophys. Acta 1226, 37–43.PubMedGoogle Scholar
  15. 15.
    Melov, S., Shoffner, J. M., Kaufman, A., and Wallace, D. C. (1995) Marked increase in the number and variety of mitochondrial DNA rearrangements in aging human skeletal muscle. Nucleic Acids Res. 23, 4122–4126.PubMedCrossRefGoogle Scholar
  16. 16.
    Munscher, C., Rieger, T., Muller-Hocker, J., and Kadenbach, B. (1993) The point mutation of mitochondrial DNA characteristic for MERRF disease is found also in healthy people of different ages. FEBS Lett. 317, 27–30.PubMedCrossRefGoogle Scholar
  17. 17.
    Palotti, F., Chen, X., Bonilla, E., and Schon, E. A. (1996) Evidence that specific mtDNA point mutations may not accumulate in skeletal muscle during normal human aging. Am. J. Hum. Genet. 59, 591–602.Google Scholar
  18. 18.
    Muller-Hocker, J. (1990) Cytochrome c oxidase deficient fibres in the limb muscle and diaphragm of man without muscular disease: an age-related alteration. J. Neurol. Sci. 100, 14–21.PubMedCrossRefGoogle Scholar
  19. 19.
    Brierley, E. J., Johnson, M. A., James, O. F. W., and Turnbull, D. M. (1996) Effects of physical activity and age on mitochondrial function. Q. J. Med. 89, 251–258.Google Scholar
  20. 20.
    Boulet, L., Karpati, G., and Shoubridge, E. A. (1992) Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF) Am. J. Hum. Genet. 51, 1187–1200.PubMedGoogle Scholar
  21. 21.
    Sciacco, M., Bonilla, E., Schon, E. A., DiMauro, S., and Moraes, C. T. (1994) Distribution of wild-type and common deletion forms of mtDNA in normal and respiration-deficient muscle fibers from pateints with mitochondrial myopathy. Hum. Mol. Genet. 3, 13–19.PubMedCrossRefGoogle Scholar
  22. 22.
    Moslemi, A.-R., Melberg, A., Holme, E., and Oldfors, A. (1996) Clonal expansion of mitochondrial DNA with multiple deletions in autosomal dominant progressive external ophthalmoplegia. Ann. Neurol. 40, 707–713.PubMedCrossRefGoogle Scholar
  23. 23.
    Brierley, E. J., Johnson, M. A., Lightowlers, R. N., James, O. F. W., and Turnbull, D. M. (1998) Role of mitochondrial DNA mutations in human aging: implications for the central nervous system and muscle. Ann. Neurol. 43, 217–223.PubMedCrossRefGoogle Scholar
  24. 24.
    Fromenty, B., Manfredi, G., Sadlock, J., Zhang, L., King, M. P., and Schon, E. (1996) Rapid mapping of identified partial duplications of human mitochondrial DNA by long PCR. Biochem. Biophys. Acta 1308, 222–230.PubMedGoogle Scholar
  25. 25.
    Reynier, P. and Malthiery, Y. (1995) Accumulation of deletions in mtDNA during tissue aging: analysis by long PCR. Biochem. Biophys. Res. Commun. 217, 59–67.PubMedCrossRefGoogle Scholar
  26. 26.
    Kovalenko, S. A., Kopsidas, G., Kelso, J. M., and Linnane, A. W. (1997) Deltoid human muscle is extensively rearranged in old age subjects. Biochem. Biophys. Res. Commun. 232, 147–152.PubMedCrossRefGoogle Scholar
  27. 27.
    De Coo, J. F. M., Gussinklo, T., Arts, P. J. W., Van Oost, B. A., and Smeets, H. J. M. (1997) A PCR test for progressive external ophthalmoplegia and Kearne-Sayre syndrome on DNA from blood samples. J. Neurol. Sci. 149, 37–40.PubMedCrossRefGoogle Scholar
  28. 28.
    Paul, R., Santucci, S., Sauntieres, A., Desnuelle, C., and Paquis-Flucklinger, V. (1996) Rapid mapping of mitochondrial DNA deletions by large fragment PCR. Trends Genet. 12, 131–132.PubMedGoogle Scholar
  29. 29.
    Johnson, M. A., Bindoff, L. A., and Turnbull, D. M. (1993) Cytochrome c oxidase activity in single muscle fibers: assay techniques and diagnostic applications. Ann. Neurol. 33, 28–35.PubMedCrossRefGoogle Scholar
  30. 30.
    Zhou, L. A., Chomyn, A., Attardi, G., and Miller, C. A. (1997) Myoclonic epilepsy and ragged red fibers (MERRF) syndrome: selective vulnerability of CNS neurons does not correlate with the level of mitochondrial tRNAlys mutation in individual neuronal isolates. J. Neurosci. 17, 7746–7753.PubMedGoogle Scholar
  31. 31.
    Ozawa, T., Tanaka, M., Sugiyama, S., Hattori, K., Ito, T., Ohno, K., Takahashi, A., Sato, W., Takada, G., Mayumi, B., Yamamoto, K., Adachi, K., Koga, Y., and Toshima, H. (1990) Multiple mitochondrial DNA deletions exist in cardiomyocytes of patients with hypertrophic or dilated cardiomyopathy. Biochem. Biophys. Res. Commun. 170, 830–836.PubMedCrossRefGoogle Scholar
  32. 32.
    Oldfors, A., Moslemi, A.-R., Fyhr, I.-M., Holme, E., Larsson, N.-G., and Lindberg, C. (1995) Mitochondrial DNA deletions in muscle fibres in inclusion body myositis. J. Neuropathol. Exp. Neurol. 54, 581–587.PubMedCrossRefGoogle Scholar
  33. 33.
    Santorelli, F. M., Sciacco, M., Tanji, K., Shanske, S., Vu, T. H., Golzi, V., Griggs, R. C., Mendell, J. R., Hays, A. P., Bertorini, T. E., Pestronk, A., Bonilla, E., and DiMauro, S. (1996) Multiple mitochondrial DNA deletions in sporadic inclusion body myositis: a study in 56 patients. Ann. Neurol. 39, 789–795.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Robert W. Taylor
    • 1
  • Theresa M. Wardell
    • 1
  • Emma L. Blakely
    • 1
  • Gillian M. Borthwick
    • 1
  • Elizabeth J. Brierley
    • 2
  • Douglass M. Turnbull
    • 1
  1. 1.Department of NeurologyThe Medical School, University of Newcastle upon TyneNewcastle upon TyneUK
  2. 2.Departments of Neurology and Geriatric MedicineThe Medical School, University of Newcastle upon TyneNewcastle upon TyneUK

Personalised recommendations