Mitochondrial aging is accelerated by anti-retroviral therapy through the clonal expansion of mtDNA mutations

Journal name:
Nature Genetics
Year published:
Published online

There is emerging evidence that people with successfully treated HIV infection age prematurely, leading to progressive multi-organ disease1, but the reasons for this are not known. Here we show that patients treated with commonly used nucleoside analog anti-retroviral drugs progressively accumulate somatic mitochondrial DNA (mtDNA) mutations, mirroring those seen much later in life caused by normal aging2, 3. Ultra-deep re-sequencing by synthesis, combined with single-cell analyses, suggests that the increase in somatic mutation is not caused by increased mutagenesis but might instead be caused by accelerated mtDNA turnover. This leads to the clonal expansion of preexisting age-related somatic mtDNA mutations and a biochemical defect that can affect up to 10% of cells. These observations add weight to the role of somatic mtDNA mutations in the aging process and raise the specter of progressive iatrogenic mitochondrial genetic disease emerging over the next decade.

At a glance


  1. COX (cytochrome c oxidase) deficiency in single skeletal muscle fibers.
    Figure 1: COX (cytochrome c oxidase) deficiency in single skeletal muscle fibers.

    (a) COX histochemistry from a representative healthy control subject (HIV−) showing normal COX activity, whereas a nucleoside analog treated HIV-infected patient (HIV+/NRTI+) shows multiple COX-deficient fibers (counterstained blue by residual SDH (succinate dehydrogenase) activity). Scale bars, 100 μm. (b) COX defects observed in each subject group (HIV+/NRTI−, HIV-infected treatment-naïve subjects; each dot represents an individual patient biopsy; ≥500 fibers sampled per biopsy).

  2. Mitochondrial DNA analysis of single skeletal muscle fibers.
    Figure 2: Mitochondrial DNA analysis of single skeletal muscle fibers.

    (a) Mitochondrial DNA (mtDNA) content in individual COX (cytochrome c oxidase)-deficient muscle fibers from nucleoside analog treated HIV-infected (HIV+/NRTI+) subjects, expressed relative to mtDNA content in adjacent fibers of normal COX activity from the same subject. A few fibers show reduced mtDNA content, whereas the majority show increased content (geometric mean of 2.1-fold proliferation, maximum 21.3-fold; P < 0.001 for difference in mean mtDNA content between COX-deficient and normal fibers). (b) The majority of COX-deficient fibers (COX−) contained high percentage levels of mtDNA containing a large-scale deletion of the major arc, causing the COX defect; whereas no deleted mtDNA was detected in adjacent COX positive fibers (COX+) (P < 0.001). (c) Schematic representation of mtDNA large-scale deletion breakpoints in COX-deficient fibers from HIV+/NRTI+ patients relative to the mtDNA gene positions (transfer RNA and ribosomal RNA not shown). Each line represents an individual deleted region. OL, origin of light chain replication; OH, origin of heavy chain replication. (n = 15 fibers from four patients).

  3. Proportional level of mt.[delta]4977 'common deletion' (CD) in homogenized skeletal muscle from HIV-infected subjects.
    Figure 3: Proportional level of mt.δ4977 'common deletion' (CD) in homogenized skeletal muscle from HIV-infected subjects.

    HIV+/NRTI+, HIV-infected, nucleoside analog exposed; HIV+/NRTI−, HIV-infected, treatment-naïve. The dashed line represents the lower threshold of the assay. NRTI-treated subjects showed significantly higher mean levels of common deletion than untreated subjects (HIV+/NRTI+ (mean ± s.e.m.), −3.45 ± 0.25 log10(/mtDNA); HIV+/NRTI−, −4.56 ± 0.31 log10(/mtDNA); P = 0.012). Box and whisker plot.

  4. Ultra-deep re-sequencing by synthesis (UDS) of skeletal muscle mtDNA.
    Figure 4: Ultra-deep re-sequencing by synthesis (UDS) of skeletal muscle mtDNA.

    UDS (Roche 454 FLX GS) shows no difference in burden of low-level mtDNA point variants (exceeding 0.2% frequency) between HIV-infected nucleoside analog treated (HIV+/NRTI+, n = 8), HIV-infected treatment-naïve (HIV+/NRTI−, n = 4) and control (HIV−, n = 4) subjects in two amplicons located in mtDNA hypervariable segment 2 (MT-HV2) and mtDNA COX subunit 3 (MT-CO3). In contrast, positive control subjects with inherited POLG defects (POLG, n = 4) show an increased burden of low-level mutations compared with healthy controls in MT-HV2 (OR = 2.33, P = 0.002).

  5. Simulations of the effects of partial mitochondrial DNA (mtDNA) replication failure caused by nucleoside analog (NRTI) exposure.
    Figure 5: Simulations of the effects of partial mitochondrial DNA (mtDNA) replication failure caused by nucleoside analog (NRTI) exposure.

    Using a validated computer model of mtDNA replication based solely on experimentally derived parameters22, we incorporated a finite period of partial replication failure caused by the mtDNA chain-terminating effects of NRTI exposure9, assigning a probability of failure per mtDNA replication event. All other parameters remained constant, including the de novo mutation rate22. We simulated 2,000 cells for 80 years. (a) The amount of mtDNA depletion during the NRTI exposure period caused by 25% and 45% probability of replication failure between 20 and 30 years of age. (>50% failure led to the complete loss of mtDNA.) The range of mtDNA depletion predicted is in keeping with published in vivo data12, 23. (b) This led to a persistent increase in the frequency of COX (cytochrome c oxidase)-deficient cells through the accelerated clonal expansion of preexisting somatic mtDNA mutations. (c) Direct simulation of the effects of NRTI exposure within our study population (two different periods, 10 and 3 years, starting at age 20, of replication failure with 45% probability). The range of COX defects predicted closely fits our empiric data. (d) Late exposure (40–50 years) had a more pronounced effect than early exposure (20–30 years) (with 45% probability of replication failure) caused by the higher number of preexisting (age-related) somatic mtDNA mutations at the time of exposure.


  1. Effros, R.B. et al. Aging and infectious diseases: workshop on HIV infection and aging: what is known and future research directions. Clin. Infect. Dis. 47, 542553 (2008).
  2. Bua, E. et al. Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers. Am. J. Hum. Genet. 79, 469480 (2006).
  3. Trifunovic, A. et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417423 (2004).
  4. Brierley, E.J., Johnson, M.A., James, O.F. & Turnbull, D.M. Effects of physical activity and age on mitochondrial function. QJM 89, 251258 (1996).
  5. Desquilbet, L. et al. HIV-1 infection is associated with an earlier occurrence of a phenotype related to frailty. J. Gerontol. A Biol. Sci. Med. Sci. 62, 12791286 (2007).
  6. Oursler, K.K., Sorkin, J.D., Smith, B.A. & Katzel, L.I. Reduced aerobic capacity and physical functioning in older HIV-infected men. AIDS Res. Hum. Retroviruses 22, 11131121 (2006).
  7. Guaraldi, G. et al. Coronary aging in HIV-infected patients. Clin. Infect. Dis. 49, 17561762 (2009).
  8. Valcour, V. et al. Higher frequency of dementia in older HIV-1 individuals: the Hawaii Aging with HIV-1 Cohort. Neurology 63, 822827 (2004).
  9. Lim, S.E. & Copeland, W.C. Differential incorporation and removal of antiviral deoxynucleotides by human DNA polymerase gamma. J. Biol. Chem. 276, 2361623623 (2001).
  10. McComsey, G.A. et al. Improvements in lipoatrophy, mitochondrial DNA levels and fat apoptosis after replacing stavudine with abacavir or zidovudine. AIDS 19, 1523 (2005).
  11. Côté, H.C. et al. Changes in mitochondrial DNA as a marker of nucleoside toxicity in HIV-infected patients. N. Engl. J. Med. 346, 811820 (2002).
  12. Maagaard, A. et al. Mitochondrial (mt)DNA changes in tissue may not be reflected by depletion of mtDNA in peripheral blood mononuclear cells in HIV-infected patients. Antivir. Ther. 11, 601608 (2006).
  13. Hayashi, J. et al. Introduction of disease-related mitochondrial DNA deletions into HeLa cells lacking mitochondrial DNA results in mitochondrial dysfunction. Proc. Natl. Acad. Sci. USA 88, 1061410618 (1991).
  14. Corral-Debrinski, M. et al. Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age. Nat. Genet. 2, 324329 (1992).
  15. Brierley, E.J., Johnson, M.A., Lightowlers, R.N., James, O.F. & Turnbull, D.M. Role of mitochondrial DNA mutations in human aging: implications for the central nervous system and muscle. Ann. Neurol. 43, 217223 (1998).
  16. Fayet, G. et al. Ageing muscle: clonal expansions of mitochondrial DNA point mutations and deletions cause focal impairment of mitochondrial function. Neuromuscul. Disord. 12, 484493 (2002).
  17. Pereira, L. et al. The diversity present in 5,140 human mitochondrial genomes. Am. J. Hum. Genet. 84, 628640 (2009).
  18. Lee, H.C., Pang, C.Y., Hsu, H.S. & Wei, Y.H. Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing. Biochim. Biophys. Acta 1226, 3743 (1994).
  19. Chinnery, P.F. & Samuels, D.C. Relaxed replication of mtDNA: a model with implications for the expression of disease. Am. J. Hum. Genet. 64, 11581165 (1999).
  20. Wanrooij, S. et al. Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA. Nucleic Acids Res. 32, 30533064 (2004).
  21. Del Bo, R. et al. Remarkable infidelity of polymerase gammaA associated with mutations in POLG1 exonuclease domain. Neurology 61, 903908 (2003).
  22. Elson, J.L., Samuels, D.C., Turnbull, D.M. & Chinnery, P.F. Random intracellular drift explains the clonal expansion of mitochondrial DNA mutations with age. Am. J. Hum. Genet. 68, 802806 (2001).
  23. Cherry, C.L. et al. Tissue-specific associations between mitochondrial DNA levels and current treatment status in HIV-infected individuals. J. Acquir. Immune Defic. Syndr. 42, 435440 (2006).
  24. Smyth, K. et al. Prevalence of and risk factors for HIV-associated neuropathy in Melbourne, Australia 1993–2006. HIV Med. 8, 367373 (2007).
  25. Diaz, F. et al. Human mitochondrial DNA with large deletions repopulates organelles faster than full-length genomes under relaxed copy number control. Nucleic Acids Res. 30, 46264633 (2002).
  26. Greaves, L.C. et al. Quantification of mitochondrial DNA mutation load. Aging Cell 8, 566572 (2009).
  27. Kollberg, G. et al. Low frequency of mtDNA point mutations in patients with PEO associated with POLG1 mutations. Eur. J. Hum. Genet. 13, 463469 (2005).
  28. World Health Organization. Antiretroviral Therapy for HIV Infection in Adults and Adolescents: A Public Health Approach (2006).
  29. Durham, S.E., Samuels, D.C., Cree, L.M. & Chinnery, P.F. Normal levels of wild-type mitochondrial DNA maintain cytochrome c oxidase activity for two pathogenic mitochondrial DNA mutations but not for m.3243Aright arrowG. Am. J. Hum. Genet. 81, 189195 (2007).
  30. Shieh, D.B. et al. Mitochondrial DNA alterations in blood of the humans exposed to N,N-dimethylformamide. Chem. Biol. Interact. 165, 211219 (2007).
  31. Bender, A. et al. High levels of mitochondrial DNA deletions in substantia nigra neurons in aging and Parkinson disease. Nat. Genet. 38, 515517 (2006).
  32. Durham, S.E., Samuels, D.C. & Chinnery, P.F. Is selection required for the accumulation of somatic mitochondrial DNA mutations in post-mitotic cells? Neuromuscul. Disord. 16, 381386 (2006).
  33. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403410 (1990).
  34. Quinlan, A.R., Stewart, D.A., Stromberg, M.P. & Marth, G.T. Pyrobayes: an improved base caller for SNP discovery in pyrosequences. Nat. Methods 5, 179181 (2008).
  35. He, Y. et al. Heteroplasmic mitochondrial DNA mutations in normal and tumour cells. Nature 464, 610614 (2010).

Download references

Author information


  1. Mitochondrial Research Group, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.

    • Brendan A I Payne,
    • Ian J Wilson,
    • Charlotte A Hateley,
    • Rita Horvath,
    • Mauro Santibanez-Koref &
    • Patrick F Chinnery
  2. Department of Infection and Tropical Medicine, Royal Victoria Infirmary, Newcastle upon Tyne, UK.

    • Brendan A I Payne &
    • D Ashley Price
  3. Centre for Human Genetics Research, Vanderbilt University, Nashville, Tennessee, USA.

    • David C Samuels

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (573K)

    Supplementary Note, Supplementary Figures 1–4 and Supplementary Tables 1, 2 and 4.

  2. Supplementary Table 3 (2M)

    UDS (Roche 454 FLX GS) outputs

Additional data