Abstract
Background
Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and age-induced cell loss in multiple tissues and mammalian species. Accurate quantitation of somatic mtDNA deletion mutations could serve as an index of age-induced cell loss. Quantitation of mtDNA deletion molecules is confounded by their low abundance in tissue homogenates, the diversity of deletion breakpoints, stochastic accumulation in single cells, and mosaic distribution between cells.
Aims
Translate a pre-clinical assay to quantitate mtDNA deletions for use in human DNA samples, with technical and biological validation, and test this assay on human subjects of different ages.
Methods
We developed and validated a high-throughput droplet digital PCR assay that quantitates human mtDNA deletion frequency.
Results
Analysis of human quadriceps muscle samples from 14 male subjects demonstrated that mtDNA deletion frequency increases exponentially with age—on average, a 98-fold increase from age 20-80. Sequence analysis of amplification products confirmed the specificity of the assay for human mtDNA deletion breakpoints. Titration of synthetic mutation mixtures found a lower limit of detection of at least 0.6 parts per million. Using muscle DNA from 6-month-old mtDNA mutator mice, we measured a 6.4-fold increase in mtDNA deletion frequency (i.e., compared to wild-type mice), biologically validating the approach.
Discussion/conclusions
The exponential increase in mtDNA deletion frequency is concomitant with the known muscle fiber loss and accelerating mortality that occurs with age. The improved assay permits the accurate and sensitive quantification of deletion mutations from DNA samples and is sufficient to measure changes in mtDNA deletion mutation frequency in healthy individuals across the lifespan and, therefore, patients with suspected mitochondrial diseases.
Similar content being viewed by others
Availability of data and material
The data that support the findings of this study are available from the corresponding author upon reasonable request.
References
Kaeberlein M (2013) Longevity and aging. F1000Prime Rep 5:5. https://doi.org/10.12703/P5-5
Ferrucci L, Gonzalez-Freire M, Fabbri E et al (2020) Measuring biological aging in humans: a quest. Aging cell 19:e13080. https://doi.org/10.1111/acel.13080
Miller RA (2001) Biomarkers of aging. http://www-personal.umich.edu/~millerr/Biomarkers.htm. Accessed 14 Sep 2020
Crimmins EM, Finch CE (2016) Constant molecular aging rates vs the exponential acceleration of mortality. Proc Natl Acad Sci USA 113:1121–1123. https://doi.org/10.1073/pnas.1524017113
Oliveira MT, Pontes CB, Ciesielski GL (2020) Roles of the mitochondrial replisome in mitochondrial DNA deletion formation. Genet Mol Biol 43:e20190069. https://doi.org/10.1590/1678-4685-GMB-2019-0069
Bua E, Johnson J, Herbst A et al (2006) Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers. Am J Hum Genet 79:469–480. https://doi.org/10.1086/507132
Cheema N, Herbst A, McKenzie D et al (2015) Apoptosis and necrosis mediate skeletal muscle fiber loss in age-induced mitochondrial enzymatic abnormalities. Aging Cell 14:1085–1093. https://doi.org/10.1111/acel.12399
Wanagat J, Cao Z, Pathare P et al (2001) Mitochondrial DNA deletion mutations colocalize with segmental electron transport system abnormalities, muscle fiber atrophy, fiber splitting, and oxidative damage in sarcopenia. FASEB J 15:322–332. https://doi.org/10.1096/fj.00-0320com
Ekstrand MI, Terzioglu M, Galter D et al (2007) Progressive parkinsonism in mice with respiratory-chain-deficient dopamine neurons. Proc Natl Acad Sci USA 104:1325–1330. https://doi.org/10.1073/pnas.0605208103
McKiernan SH, Tuen VC, Baldwin K et al (2007) Adult-onset calorie restriction delays the accumulation of mitochondrial enzyme abnormalities in aging rat kidney tubular epithelial cells. Am J Physiol Renal Physiol 292:F1751–F1760. https://doi.org/10.1152/ajprenal.00307.2006
Baris OR, Ederer S, Neuhaus JF et al (2015) Mosaic deficiency in mitochondrial oxidative metabolism promotes cardiac arrhythmia during aging. Cell Metab 21:667–677. https://doi.org/10.1016/j.cmet.2015.04.005
Warner HR, Hodes RJ, Pocinki K (1997) What does cell death have to do with aging? J Am Geriatr Soc 45:1140–1146. https://doi.org/10.1111/j.1532-5415.1997.tb05981.x
Herbst A, Widjaja K, Nguy B et al (2017) Digital PCR quantitation of muscle mitochondrial DNA: age, fiber type, and mutation-induced changes. J Gerontol 72:1327–1333. https://doi.org/10.1093/gerona/glx058
Corral-Debrinski M, Horton T, Lott MT et al (1992) Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age. Nat Genet 2:324–329. https://doi.org/10.1038/ng1292-324
Wallace DC (1992) Diseases of the mitochondrial DNA. Annu Rev Biochem 61:1175–1212. https://doi.org/10.1146/annurev.bi.61.070192.005523
Yu-Wai-Man P, Lai-Cheong J, Borthwick GM et al (2010) Somatic mitochondrial DNA deletions accumulate to high levels in aging human extraocular muscles. Invest Ophthalmol Vis Sci 51:3347–3353. https://doi.org/10.1167/iovs.09-4660
Herbst A, Hoang AN, Woo W et al (2019) Mitochondrial DNA alterations in aged macrophage migration inhibitory factor-knockout mice. Mechanisms of ageing and development 182:111126. https://doi.org/10.1016/j.mad.2019.111126
Taylor SD, Ericson NG, Burton JN et al (2014) Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain. Aging Cell 13:29–38. https://doi.org/10.1111/acel.12146
Bielas J, Herbst A, Widjaja K et al (2018) Long term rapamycin treatment improves mitochondrial DNA quality in aging mice. Exp Gerontol 106:125–131. https://doi.org/10.1016/j.exger.2018.02.021
O’Hara R, Tedone E, Ludlow A et al (2019) Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution. Genome Res 29:1878–1888. https://doi.org/10.1101/gr.250480.119
Belmonte FR, Martin JL, Frescura K et al (2016) Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions. Sci Rep 6:25186. https://doi.org/10.1038/srep25186
Lee CC, Hoang A, Segovia D et al (2020) Enhanced methods for needle biopsy and cryopreservation of skeletal muscle in older adults. J Cytol Histol. https://doi.org/10.3742/jch.2020.11.553
Slichter SJ, Harker LA (1976) Preparation and storage of platelet concentrates. Transfusion 16:8–12. https://doi.org/10.1046/j.1537-2995.1976.16176130842.x
Goudenege D, Bris C, Hoffmann V et al (2019) eKLIPse: a sensitive tool for the detection and quantification of mitochondrial DNA deletions from next-generation sequencing data. Genet Med 21:1407–1416. https://doi.org/10.1038/s41436-018-0350-8
Cao Z, Wanagat J, McKiernan SH et al (2001) Mitochondrial DNA deletion mutations are concomitant with ragged red regions of individual, aged muscle fibers: analysis by laser-capture microdissection. Nucleic Acids Res 29:4502–4508. https://doi.org/10.1093/nar/29.21.4502
Shoffner JM, Lott MT, Voljavec AS et al (1989) Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip-replication model and metabolic therapy. Proc Natl Acad Sci USA 86:7952–7956. https://doi.org/10.1073/pnas.86.20.7952
Cortopassi GA, Arnheim N (1990) Detection of a specific mitochondrial DNA deletion in tissues of older humans. Nucleic acids Res 18:6927–6933. https://doi.org/10.1093/nar/18.23.6927
Lee CM, Chung SS, Kaczkowski JM et al (1993) Multiple mitochondrial DNA deletions associated with age in skeletal muscle of rhesus monkeys. J Gerontol 48:B201–B205. https://doi.org/10.1093/geronj/48.6.b201
Vermulst M, Wanagat J, Kujoth GC et al (2008) DNA deletions and clonal mutations drive premature aging in mitochondrial mutator mice. Nat Genet 40:392–394. https://doi.org/10.1038/ng.95
Bua EA, McKiernan SH, Wanagat J et al (2002) Mitochondrial abnormalities are more frequent in muscles undergoing sarcopenia. J Appl Physiol (1985) 92:2617–2624. https://doi.org/10.1152/japplphysiol.01102.2001
Wanagat J, Wolff MR, Aiken JM (2002) Age-associated changes in function, structure and mitochondrial genetic and enzymatic abnormalities in the Fischer 344 x Brown Norway F(1) hybrid rat heart. J Mol Cell Cardiol 34:17–28. https://doi.org/10.1006/jmcc.2001.1483
Pak JW, Herbst A, Bua E et al (2003) Mitochondrial DNA mutations as a fundamental mechanism in physiological declines associated with aging. Aging Cell 2:1–7. https://doi.org/10.1046/j.1474-9728.2003.00034.x
Someya S, Yamasoba T, Kujoth GC et al (2008) The role of mtDNA mutations in the pathogenesis of age-related hearing loss in mice carrying a mutator DNA polymerase gamma. Neurobiol Aging 29:1080–1092. https://doi.org/10.1016/j.neurobiolaging.2007.01.014
Lexell J, Taylor CC, Sjostrom M (1988) What is the cause of the ageing atrophy? Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-year-old men. J Neurol Sci 84:275–294. https://doi.org/10.1016/0022-510x(88)90132-3
Administration SS (2015) Actuarial Life Table. https://www.ssa.gov/oact/STATS/table4c6.html
Belsky DW, Moffitt TE, Cohen AA et al (2018) Eleven telomere, epigenetic clock, and biomarker-composite quantifications of biological aging: do they measure the same thing? Am J Epidemiol 187:1220–1230. https://doi.org/10.1093/aje/kwx346
Gompertz B (1825) On the nature of the function expressive of the law of human mortality and on a new model of determining life contingencies. Philos Trans R Soc 115:513–585
Studenski S, Perera S, Patel K et al (2011) Gait speed and survival in older adults. JAMA 305:50–58. https://doi.org/10.1001/jama.2010.1923
Hepple RT (2014) Mitochondrial involvement and impact in aging skeletal muscle. Front Aging Neurosci 6:211. https://doi.org/10.3389/fnagi.2014.00211
Lexell J, Downham D, Sjostrom M (1986) Distribution of different fibre types in human skeletal muscles. Fibre type arrangement in m. vastus lateralis from three groups of healthy men between 15 and 83 years. J Neurol Sci 72:211–222. https://doi.org/10.1016/0022-510x(86)90009-2
Herbst A, Pak JW, McKenzie D et al (2007) Accumulation of mitochondrial DNA deletion mutations in aged muscle fibers: evidence for a causal role in muscle fiber loss. J Gerontol 62:235–245. https://doi.org/10.1093/gerona/62.3.235
Gokey NG, Cao Z, Pak JW et al (2004) Molecular analyses of mtDNA deletion mutations in microdissected skeletal muscle fibers from aged rhesus monkeys. Aging Cell 3:319–326. https://doi.org/10.1111/j.1474-9728.2004.00122.x
Hazkani-Covo E, Zeller RM, Martin W (2010) Molecular poltergeists: mitochondrial DNA copies (numts) in sequenced nuclear genomes. PLoS Genet 6:e1000834. https://doi.org/10.1371/journal.pgen.1000834
Parikh S, Karaa A, Goldstein A et al (2019) Diagnosis of ‘possible’ mitochondrial disease: an existential crisis. J Med Genet 56:123–130. https://doi.org/10.1136/jmedgenet-2018-105800
Rahman S (2020) Mitochondrial disease in children. J Intern Med 286:609–633. https://doi.org/10.1111/joim.13054
Ahmed ST, Craven L, Russell OM et al (2018) Diagnosis and treatment of mitochondrial myopathies. Neurotherapeutics 15:943–953. https://doi.org/10.1007/s13311-018-00674-4
Bioanalytical Method Validation Guidance for Industry (2018)
Funding
This work is supported by the National Institute on Aging at the National Institutes of Health (Grant numbers R56AG060880, R01AG055518, K02AG059847, and R21AR072950).
Author information
Authors and Affiliations
Contributions
AH, JMA, DM and JW conceived and planned the experiments. CCL provided the de-identified muscle samples. AH purified DNA and performed all digital PCR measurements. AH, NL, DA, AV and JW analyzed the data. JW and AH wrote the manuscript with input from all authors.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflicts of interest/competing interests.
Ethics approval (include appropriate approvals or waivers)
Animal studies were conducted according to the Guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research and were approved by the UCLA Institutional Animal Care and Use Committee. De-identified muscle biopsy specimens were collected as part of a VA Merit Award, “Testosterone, inflammation and metabolic risk in older Veterans” and NIH R01DK090406 (PI: Cathy Lee, MD). Use of the human specimens for this study was approved by the UCLA Institutional Review Board (Protocol #18-001547) and the University of Alberta Health Research Ethics Board #00084515.
Consent to participate
This study involved the use of de-identified human muscle biopsies and therefore did not include human subjects.
Consent for publication
This study involved the use of de-identified human muscle biopsies and therefore did not include human subjects.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Herbst, A., Lee, C.C., Vandiver, A.R. et al. Mitochondrial DNA deletion mutations increase exponentially with age in human skeletal muscle. Aging Clin Exp Res 33, 1811–1820 (2021). https://doi.org/10.1007/s40520-020-01698-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s40520-020-01698-7