Mitochondrial DNA deletion mutations increase exponentially with age in human skeletal muscle
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ORIGINAL ARTICLE
Mitochondrial DNA deletion mutations increase exponentially with age in human skeletal muscle Allen Herbst1 · Cathy C. Lee2,3 · Amy R. Vandiver4 · Judd M. Aiken1 · Debbie McKenzie5 · Austin Hoang3 · David Allison6 · Nianjun Liu6 · Jonathan Wanagat2,3 Received: 8 June 2020 / Accepted: 26 August 2020 © Springer Nature Switzerland AG 2020
Abstract Background Mitochondrial DNA (mtDNA) deletion mutations lead to electron transport chain-deficient cells and ageinduced 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. Keywords MtDNA · Mitochondria · Mutation · Deletion · Sarcopenia · Biomarker
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s40520-020-01698-7) contains supplementary material, which is available to authorized users. * Jonathan Wanagat [email protected] 1
Age is the primary risk factor for the leading causes of disability and death [1]. While aging has traditionally been classified as a non-modifiable risk factor, this is no longer 4
Division of Dermatology, Department of Medicine, UCLA, 10945 Le Conte Ave, Suite 2339, Los Angeles, CA 90095, USA
Department of Agricultural, Food and Nutritional Sciences, University of Alberta, Edmonton, AB, Canada
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Veterans Administration Greater Los Angeles Hea
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