Ultra-Sensitive Sequencing Reveals an Age-Related
`Increase in Somatic Mitochondrial Mutations That Are
`Inconsistent with Oxidative Damage
`
`Scott R. Kennedy1, Jesse J. Salk1,2, Michael W. Schmitt1,2, Lawrence A. Loeb1,3*
`
`1 Department of Pathology, University of Washington, Seattle, Washington, United States of America, 2 Department of Medicine, University of Washington, Seattle,
`Washington, United States of America, 3 Department of Biochemistry, University of Washington, Seattle, Washington, United States of America
`
`Abstract
`
`Mitochondrial DNA (mtDNA) is believed to be highly vulnerable to age-associated damage and mutagenesis by reactive
`oxygen species (ROS). However, somatic mtDNA mutations have historically been difficult to study because of technical
`limitations in accurately quantifying rare mtDNA mutations. We have applied the highly sensitive Duplex Sequencing
`methodology, which can detect a single mutation among .107 wild type molecules, to sequence mtDNA purified from
`human brain tissue from both young and old individuals with unprecedented accuracy. We find that the frequency of point
`mutations increases ,5-fold over the course of 80 years of life. Overall, the mutation spectra of both groups are comprised
`predominantly of transition mutations, consistent with misincorporation by DNA polymerase c or deamination of cytidine
`and adenosine as the primary mutagenic events in mtDNA. Surprisingly, GRT mutations, considered the hallmark of
`oxidative damage to DNA, do not significantly increase with age. We observe a non-uniform, age-independent distribution
`of mutations in mtDNA, with the D-loop exhibiting a significantly higher mutation frequency than the rest of the genome.
`The coding regions, but not the D-loop, exhibit a pronounced asymmetric accumulation of mutations between the two
`strands, with GRA and TRC mutations occurring more often on the light strand than the heavy strand. The patterns and
`biases we observe in our data closely mirror the mutational spectrum which has been reported in studies of human
`populations and closely related species. Overall our results argue against oxidative damage being a major driver of aging
`and suggest that replication errors by DNA polymerase c and/or spontaneous base hydrolysis are responsible for the bulk of
`accumulating point mutations in mtDNA.
`
`Citation: Kennedy SR, Salk JJ, Schmitt MW, Loeb LA (2013) Ultra-Sensitive Sequencing Reveals an Age-Related Increase in Somatic Mitochondrial Mutations That
`Are Inconsistent with Oxidative Damage. PLoS Genet 9(9): e1003794. doi:10.1371/journal.pgen.1003794
`
`Editor: Bennett Van Houten, University of Pittsburgh, United States of America
`
`Received April 22, 2013; Accepted July 29, 2013; Published September 26, 2013
`Copyright: ß 2013 Kennedy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
`unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
`
`Funding: Support for this research was provided by the following grants: NIA P01-AG01751, NCI PO1-CA77852, and NCI RO1-CA102029 to LAL. SRK was further
`supported by the Genetic Approaches to Aging Training Grant (NIA T32-AG000057). Tissues were collected by the University of Washington Alzheimer’s Disease
`Research Center (P50AG05136). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
`
`Competing Interests: The authors have declared that no competing interests exist.
`
`* E-mail: laloeb@u.washington.edu
`
`Introduction
`
`Mitochondria are the primary source of energy for cells.
`Owing to their evolutionary history, these organelles harbor a
`small,
`independently replicated genome (mtDNA). Human
`mtDNA encodes two rRNA genes, 13 protein coding genes that
`are essential components of the electron transport chain (ETC),
`and a full complement of 22 tRNAs used in translation of the
`ETC peptides. The escape of electrons from the ETC can lead to
`the formation of reactive oxygen species (ROS), which are
`capable of damaging a variety of cellular components, including
`DNA. Due to its proximity to the ETC, absence of protective
`histones, and a lack of nucleotide excision or mismatch repair,
`mtDNA is thought to be especially vulnerable to ROS-mediated
`damage and the generation of mutations. Failure to faithfully
`transmit
`the encoded information during mtDNA replication
`leads to the production of dysfunctional ETC proteins, leading to
`the release of more free electrons and ROS in what has been
`termed ‘the vicious cycle’ [1,2]. Thus, it is not surprising that
`mutations in mtDNA have been associated with a decline in
`energy production, a loss of organismal
`fitness, an increased
`
`propensity for a number of pathological conditions, and aging
`(reviewed in [3,4]).
`Numerous lines of evidence have suggested mtDNA mutations
`are involved in the aging process. In particular, ETC activity
`declines with age [5,6], and this decrease is coincident with
`accumulation of mitochondria with large deletions
`in their
`mtDNA [7,8,9,10]. Large, kilobase-sized deletions in mtDNA
`become more prevalent with age in a variety of tissues, including
`brain [11], heart [12], and skeletal muscle [7]. Furthermore, these
`large deletions have been shown to increase in frequency in a
`number of neurodegenerative conditions,
`including Parkinson’s
`disease [13,14] and Alzheimer’s disease [15]. In addition, DNA
`damage, predominantly in the form of 8-hydroxy-29-deoxyguano-
`sine (8-oxo-dG) [16],
`increases with age in both nuclear and
`mitochondrial DNA [17,18,19,20]. While the role of mtDNA
`deletions in aging is well established, the role of point mutations
`remains controversial [21,22].
`Several previous studies have examined the accumulation of
`point mutations in human aging and disease [23,24,25,26]. Until
`very recently, hypotheses that required the observation of rare
`mutations in mtDNA have been extremely difficult to experimen-
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`Author Summary
`
`Owing to their evolutionary history, mitochondria harbor
`independently replicating genomes. Failure to faithfully
`transmit
`the genetic information of mtDNA during
`replication can lead to the production of dysfunctional
`electron transport proteins and a subsequent decline in
`energy production. Cellularly-derived reactive oxygen
`species (ROS) and environmental agents preferentially
`damage mtDNA compared to nuclear DNA. However, little
`is known about the consequences of mtDNA damage for
`mutagenesis. This lack of knowledge stems, in part, from
`an absence of methods capable of accurately detecting
`these mutations throughout the mitochondrial genome.
`Using a new, highly sensitive DNA sequencing strategy, we
`find that the frequency of point mutations is 10–100-fold
`lower than what has been previously reported using less
`precise means. Moreover, the frequency increases 5-fold
`over an 80 year
`lifespan. We also find that
`it
`is
`predominantly transition mutations, rather than mutations
`commonly associated with oxidative damage to mtDNA,
`that increase with age. This finding is inconsistent with free
`radical theories of aging. Finally, the mutagenic patterns
`and biases we observe in our data are similar to what is
`seen in population studies of mitochondrial polymor-
`phisms and suggest a common mechanism by which
`somatic and germline mtDNA mutations arise.
`
`tally validate due to: 1) the lack of genetic tools for introducing
`reporters or
`selectable markers
`into mtDNA; 2)
`the high
`background error
`rate of most DNA sequencing methods
`[27,28]; and 3) the sampling limitations of the few available
`high-sensitivity mutation assays that screen only a tiny subset of
`the genome [29]. The mitochondrial genome is 16,569 bp, and
`individual human cells frequently contain hundreds to thousands
`of molecules of mtDNA;
`thus, a single human cell
`typically
`contains millions of nucleotides of mtDNA sequence. The rate of
`accumulation of mtDNA mutations has previously been estimated
`as 661028 mutations per base pair per year [30]. Therefore,
`reliable study of spontaneous mtDNA mutations requires meth-
`odologies that can accurately detect a single mutation among
`.106 wild-type base-pairs. However, most prior studies of mtDNA
`mutations and aging have relied upon methods with background
`error frequencies of 1023 to 1024; hence the many reported
`differences likely reflect changes in mutation clonality or technical
`artifacts (e.g. due to increases in DNA damage with age) rather
`than true spontaneous mutations.
`Massively parallel sequencing technologies allow mtDNA to be
`subjected to ‘deep sequencing’ in order to detect rare/sub-clonal
`mutations on a genome-wide level. However, these new sequenc-
`ing methods are highly error prone, with artifactual error rates of
`approximately one spurious mutation per 100 to 1,000 nucleotides
`sequenced. These high error rates have precluded the study of
`spontaneous mutations in mtDNA [31]. To circumvent
`this
`limitation, we recently developed a new, highly accurate
`sequencing methodology termed Duplex Sequencing (DS), which
`has the unique ability to detect a single mutation among .107
`sequenced bases [32].
`In the study herein, we determined the effect of aging on
`mtDNA mutation burden by using DS to compare human
`mtDNA purified from brain tissue of five young individuals (ages
`,1) and five aged individuals (ages 75–99 years) obtained via
`rapid autopsy (Table S1). As brain is among the most
`metabolically active tissues in the human body, we reasoned it
`
`Somatic Mitochondrial Mutations Increase with Age
`
`to be particularly prone to damage from ROS, and thus, an
`optimal tissue for comparison between age groups. We assessed
`the relative frequency, spectrum, and distribution of mtDNA
`mutations in the two groups. We find that point mutations
`increase with age, but do so in a non-uniform manner.
`Furthermore, we find that mutations
`show a bias
`in their
`occurrence with respect to both genome location and strand
`orientation. The types of mutations we detect are inconsistent
`with oxidative damage being a major driver of mtDNA
`mutagenesis.
`
`Results
`
`Duplex Sequencing relies on the concept of molecular tagging
`and the fact that the two strands of DNA contain complementary
`information. Fragmented duplex DNA is tagged with adapters
`bearing a random, yet complementary, double-stranded nucleo-
`tide sequence (Fig. 1A). Following ligation,
`the individually
`labeled strands are PCR amplified,
`thus creating sequence
`‘‘families’’ that share a common tag sequence, derived from
`each of
`the two single parental
`strands
`(Fig. 1B). After
`sequencing, members of each duplicate family are grouped by
`tag, and a consensus sequence is calculated for each family,
`creating a single strand consensus sequence (SSCS)
`(Fig. 1C).
`This step eliminates random sequencing or PCR errors that occur
`during library amplification; however, the single-stranded con-
`sensus process does not filter out artifactual mutations that are the
`consequence of first round PCR errors, such as those commonly
`caused by DNA damage. To remove this latter type of error, the
`complementary SSCS families derived from the two single-
`stranded halves of the original DNA duplex are compared to
`each other (Fig. 1C). The base identity at each position in a read
`is kept in the final consensus only if the two strands match
`perfectly at
`that position. Upon remapping of
`these duplex
`consensus sequence (DCS) reads back to the reference genome,
`any deviations from the reference sequence are considered true
`mutations. The frequency of mutations in a sampled population
`of mtDNA is calculated as the number of DCS mutant molecules
`divided by the number of DCS wild-type molecules observed at
`any given genomic position.
`
`Point mutations accumulate with aging in human
`mtDNA
`Point mutations in mtDNA could be the result of maternal
`inheritance or a de novo mutation event. Maternally inherited
`mutations or mutations arising during early embryonic develop-
`ment are more likely to be clonal (i.e. the same mutation being
`present at the same location in most or all mtDNA molecules).
`Therefore, in order to quantify the frequency of de novo events, we
`used a clonality cutoff that excluded any positions with variants
`occurring at a frequency of .1%, and scored each type of
`mutation only once at each position of the genome. Based on these
`criteria,
`the mtDNA from aged individuals
`show a highly
`significant ,5-fold increase in mutational frequency, relative to
`those obtained from young individuals (Young: 3.760.961026 vs.
`Aged: 1.960.261025, p,1024, two-sample t-test) (Fig. 2A). These
`mutation frequencies are between one and two orders of
`magnitude lower than the previously reported values for both
`young and old individuals using PCR-based methods or conven-
`tional next-generation sequencing [24,33,34]. This discordance
`likely stems from artifactual scoring of mutations by these latter
`methods due to misinsertion of incorrect bases at sites of damage
`in template DNA during the PCR steps. Duplex Sequencing, in
`contrast, is unaffected by DNA damage [32].
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`Somatic Mitochondrial Mutations Increase with Age
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`Figure 1. Overview of the Duplex Sequencing methodology. (A) Adapter design with random double-stranded tag sequence and invariant
`spacer sequence. (B) Ligation of adapters to fragmented DNA generates unique 12 bp tags on each end (a and b). PCR amplification of the two
`strands produces two related, but distinct products. (C) Sequence reads sharing unique a and b tags are grouped into families of a-b or b-a
`orientation. Mutations are of three different types: sequencing mistakes or late arising PCR errors (blue or purple spots); first round PCR errors (brown
`spots); true mutations (green spots). Comparing SSCSs from the paired families generates a DCS, which eliminates all but true mutations.
`doi:10.1371/journal.pgen.1003794.g001
`
`Inspection of the mutation spectra for both the young and old
`samples reveals that all samples are significantly biased towards
`transitions (Fig. 2B). Specifically, the most common mutation type,
`GRA/CRT, is consistent with either misincorporation by DNA
`polymerase c or deamination of cytosine to form uracil, as being
`the largest mutagenic drivers in mtDNA [35,36]. The second most
`common mutation type, TRC/ARG, is consistent with either
`deamination of adenosine to inosine or a T-dGTP mispairing, the
`primary base misinsertion mistake made by DNA polymerase c
`[37,38,39,40]. Plotting the frequency of each type of mutation as a
`proportion of total mutations (Fig. 2C) reveals that the relative
`abundance of each mutation type is the same in young and old,
`suggesting that the mutagenic pressures that result in the observed
`spectra are constant throughout the human lifespan.
`Surprisingly, comparison of the mutation spectra of the young
`and old samples reveals a notable absence of the mutational
`signature of oxidative damage. A number of studies have shown
`that oxidative damage to DNA accumulates in both the nuclear
`and mitochondrial genomes as a function of age, as well as several
`age-associated pathologies [17,18,19,20,41]. The most frequent
`alteration produced by oxidative damage is 8-oxo-dG, which,
`when copied during replication or repair, results in dA substitu-
`tions, yielding GRT/CRA transversions [42]. A number of
`theories of aging invoke ROS-mediated damage to mtDNA as
`being a major driver of the aging phenotype (reviewed in [43] and
`[44]). A key prediction for these theories is that the frequency of
`GRT/CRA mutations would be expected to increase with time.
`We failed to find either a preponderance of GRT/CRA
`substitutions or a proportionally greater increase with age in this
`type of mutation relative to other types, despite a span of .80
`years between our sequenced sample groups (Fig. 2C).
`
`Deleterious mutations increase with age
`Our data indicate that point mutations increase with age and
`that these mutations are inconsistent with oxidative damage being
`a primary driver of mutagenesis; we next assessed whether these
`mutations lead to alterations in the protein coding sequence. We
`find that in the aged samples, 78.3% of mutations are non-
`synonymous. The incidence of non-synonymous mutations is close
`to the expected value of 75.7% for mtDNA that would occur if
`non-synonymous and synonymous mutations occur randomly. In
`contrast, only 62.9% of mutations are non-synonymous in the
`young samples. The reduced mutation load observed in the young
`samples is consistent with that negative intergenerational selection
`against such mutations and that this selection is relieved during
`development and could play a role in the aging phenotype.
`However, the existence of a high load of non-synonymous
`mutations does not necessarily mean that the coding changes lead
`to functional protein alteration. To examine this possibility, we
`compared the predicted ‘‘pathogenicity’’ of all non-synonymous
`mutations in both the young and aged samples using MutPred
`[45], a software package that calculates the likelihood of a
`mutation being deleterious based on a number of criteria,
`including protein structure, the presence of
`functional protein
`motifs, evolutionary conservation, and amino acid composition
`bias. A score between zero and one is assigned to each mutation,
`with a higher score denoting a higher likelihood of being
`deleterious. Based on this analysis
`(Fig. S1),
`the predicted
`pathogenicity of mutations, indeed, increases with age (p,0.02,
`Wilcoxon Rank Sum analysis), suggesting that mutations acquired
`during aging may have functional consequences for the electron
`transport chain. A similar increase in predicted deleterious
`mutations was also observed using the SWIFT software package
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`Somatic Mitochondrial Mutations Increase with Age
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`Figure 2. Mitochondrial point mutations increase with age and
`are biased to transitions. (A) mtDNA point mutations burden is
`higher in older individuals (purple) than young individuals (yellow)
`(p,1024, two-tailed t-test). Error bars represent the 95% confidence
`interval for each sample (Wilson Score interval) (B) The mutation spectra
`of both young (yellow) and aged (purple) individuals shows an excess of
`transitions, relative to transversions. Frequencies were calculated by
`dividing the number of mutations of each type by the number of times
`the wild-type base of each mutation type was sequenced. Indels were
`calculated independently as events per
`total number of bases
`sequenced. Error bars represent one standard deviation.
`(C) The
`mutation spectra, reported as the relative proportion of the different
`mutation types, do not change with age. Error bars represent one
`standard deviation. Significance was tested using the two-tailed t-test.
`doi:10.1371/journal.pgen.1003794.g002
`
`(data not shown). The increase in predicted pathogenicity is
`consistent with mutations causing coding changes occurring
`randomly and argues against a mechanism by which point
`mutations are selected against by the cell. Similar finding in
`clonally expanded mutations were recently reported in colon tissue
`show a similar increase in predicted pathogenic mutation in
`mtDNA [46].
`
`The D-loop of mtDNA exhibits an elevated mutation
`burden but is not a mutagenic ‘hotspot’
`The mitochondrial genome can be divided into three different
`regions: 1) protein coding genes, 2) RNA coding genes (consisting
`of both rRNA and tRNA), and 3) non-coding/regulatory regions
`including the origin of
`replication known as
`the D-loop.
`Phylogenetic analysis of both human and other mammalian
`lineages has shown that population level single nucleotide variants
`(SNVs)
`tend to cluster
`in a number of
`‘hotspots’
`in the
`mitochondrial genome, most notably in Hypervariable Regions I
`and II of the D-loop [47,48,49]. We sought to determine if the
`distribution of non-clonal mutations within the mtDNA of
`individuals exhibited a uniform distribution or if certain regions
`of the genome similarly show variations in mutation frequency.
`Comparison of the mutation frequencies of the RNA coding genes
`to the protein coding genes yielded no significant differences in
`either the young or old samples (p = 0.15, two-tailed t-test).
`In contrast, we observed a significant increase in mutation
`frequency of the D-loop (bp 16024-576) relative to the coding
`regions (bp 577–16023) in both young (D-loop: 1.560.661025 vs.
`coding region: 2.960.761026, p,0.01, two-tailed t-test) and aged
`(D-loop: 5.761.561025 vs. coding region: 1.6560.261025,
`p,0.01, two-tailed t-test) samples, suggesting that the D-loop is
`a mutagenic hotspot. However comparing the relative increase in
`the mutation frequency of the D-loop between the young and old
`sample groups (3.861.6-fold increase) to the relative increase seen
`between the two sample groups
`in the non-D-loop regions
`(5.662.0 fold increase) shows no difference. This finding is
`inconsistent with the idea that the D-loop accumulates significantly
`more mutations during aging than the rest of the mitochondrial
`genome. Spectrum analysis shows a similar predominance of
`transition mutations in both the D-loop and coding regions of the
`genome (Fig. 3A), with no significant difference in the relative
`abundance of
`the different mutation types (Fig. 3B). Taken
`together, our data suggest that the mutagenic processes of mtDNA
`are largely uniform across the genome.
`
`Mutations accumulate asymmetrically on the two strands
`of mtDNA
`The human mitochondrial genome has a significant bias in the
`cytosine/guanine composition between the two strands. Specifi-
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`Somatic Mitochondrial Mutations Increase with Age
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`hypothesized to be due to an asymmetric accumulation of
`mutations between the two strands of mtDNA in the germline;
`however, to date, the biases have not been observed at the sub-
`clonal/random level within individuals. To examine this, we
`compared the frequency of reciprocal mutations occurring on the
`L-strand (i.e. GRA on the L-strand vs. CRT mutations on the L-
`strand). By definition, mutations cause complementary sequence
`changes on both strands of a DNA molecule. Therefore, if a bias
`does not exist in the orientation of specific mutations towards a
`particular strand, then the frequency of reciprocal mutations on
`the same strand would be expected to be equal. Alternatively, the
`presence of a strand orientation bias would manifest itself in the
`form of a particular type of mutation occurring more frequently
`than its reciprocal mutation.
`We find that the majority of the human mitochondrial genome
`shows a significant strand orientation bias in the occurrence of
`transitions, whereas transversions show no apparent asymmetry
`(Fig. 4A). Specifically, in young samples, GRA/CRT mutations
`are more likely to occur when the dG base is present on the L-
`strand and the dC base is in the H-strand, respectively. This
`pattern is even more pronounced in aged individuals, consistent
`with this bias being due to ongoing mutagenic process and not the
`result of maternal inheritance. In addition to the GRA/CRT
`bias, the aged samples also exhibit a strand orientation bias in the
`occurrence of TRC/ARG, where dT is more likely to be
`mutated to a dC when it is located on the L-strand than on the H-
`strand. Interestingly, this bias, which appears uniformly through-
`out most of the mtDNA, is uniquely absent in the D-loop region
`(Fig. 4B). Thus, both the spectrum and strand orientation
`asymmetry of somatic mtDNA mutation accumulation recapitu-
`lates what has been previously recognized in population studies.
`
`Discussion
`
`in mtDNA has
`somatic mutations
`The accumulation of
`frequently been hypothesized to drive the aging process and its
`associated pathologies, including neurodegeneration, cancer, and
`atrophy (reviewed in [54]). The underlying mechanisms by which
`these mutations occur and accumulate have been the subject of
`intense study, but remain incompletely defined. One of the major
`limitations has been the lack of methodologies with sufficient
`sensitivity to detect
`rare mutations among a much larger
`population of wild-type molecules. We recently developed a
`robust next-generation sequencing methodology, termed Duplex
`Sequencing, which is able to detect a single point mutation among
`.107 sequenced bases [32] and has now enabled us to precisely
`characterize the genome-wide frequency, spectrum, and distribu-
`tion of somatic mtDNA mutations in aging human brain with
`unprecedented accuracy.
`Our data show a significant increase in the load of point
`mutations as a function of human age, with absolute frequencies
`10–100 fold lower than what has been typically reported in the
`literature using less
`sensitive assays. Recent work using the
`Random Mutation Capture assay has reported an age associated
`increase in mtDNA point mutation frequencies in mice and
`Drosophila that are on par with the values that we have determined
`here; however, these studies were limited to only a very small
`region of the genome [22] (Leo Pallanck-submitted). Of particular
`interest, despite a ,1000-fold difference in lifespan, the increase in
`mutation load with age appears to be highly consistent among
`multiple species. This
`surprising finding suggests
`that
`the
`underlying mechanisms behind the age-dependent accumulation
`of point mutations in mtDNA are conserved between humans,
`flies, and mice and merit more detailed comparison.
`
`Figure 3. The D-loop has an elevated mutation burden but its
`mutation spectrum is similar to the remainder of the mito-
`(A) The D-loop (orange) exhibits a higher
`chondrial genome.
`aggregate mutation burden than the rest of the genome (grey).
`Frequencies were calculated by dividing the number of mutations of
`each type by the number of times the wild-type base of each mutation
`type was sequenced. Indels were calculated independently as events
`per total number of bases sequenced. Error bars represent one standard
`deviation. (B) The relative fraction of mutations exhibits no difference
`between the D-loop (orange) and non-D-loop (grey) portions of the
`genome, suggesting a similar underlying mutagenic process. Error bars
`represent one standard deviation.
`doi:10.1371/journal.pgen.1003794.g003
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`cally, the light strand (L-strand), which is the coding strand for
`only nine genes, contains about three-fold more cytosine than
`guanine, whereas the heavy strand (H-strand) codes for the
`remaining 28 genes and has the opposite composition bias.
`Human population studies, as well as the comparative analysis of
`evolutionarily related species, have shown a bias towards the
`occurrence of GRA and TRC SNPs of
`the L-strand
`[50,51,52,53]. These population-level compositional biases are
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`Figure 4. Transition mutations show a pronounced strand bias in their occurrence. (A) Comparison of frequencies of reciprocal mutation
`types on the L-strand for both young (yellow) and aged (purple) sample groups shows that young individuals exhibit a strand bias in the occurrence
`of GRA/CRT mutations. Aged samples exhibit an even stronger bias in both GRA/CRT and TRC/ARG mutations. No strand bias was observed for
`transversion mutations. (B) Comparison of frequencies of reciprocal mutation types on the L-strand from aged individuals shows no significant strand
`bias in the D-loop (bp 16,025-576) region (grey) of mtDNA, while the non-D-loop portion (bp 577–16,024) (orange) of the genome from the same
`aged samples exhibits a strong strand bias. Frequencies were calculated by dividing the number of mutations of each type by the number times the
`wild-type base of each mutation type was sequenced. Error bars represent one standard deviation. Significance was tested using the two-tailed t-test.
`doi:10.1371/journal.pgen.1003794.g004
`
`Oxidative damage to DNA, most notably in the form of 8-oxo-
`dG, has long been believed to be a primary driver of mutagenesis
`in both nuclear and mitochondrial DNA [42,55,56]. However, our
`results do not support this hypothesis. In our data, the relative
`proportion of GRT/CRA mutations is quite low in the young
`samples examined and, importantly, does not show a dispropor-
`tionate increase with age relative to other types of mutations.
`Other recent reports, which used less sensitive methods to detect
`intermediate frequency sub-clonal mutations, have similarly failed
`to detect this classic signature of oxidative damage to DNA. For
`instance, one conventional deep sequencing analysis of aged mice
`reported no significant burden of GRT/CRA transversions [57].
`Even more surprising is the observation that a transgenic mouse
`strain deficient for both MutY and OGG1, which are the two
`primary enzymes responsible for repairing 8-oxo-dG, do not
`exhibit an increase in mtDNA mutations [58].
`
`Comparison of the spectrum of our reported data (Fig. 2B) to
`that of the clonal SNV’s in our data (i.e. mutations present at
`.90%), as well as those reported in the Mitomap database [59],
`reveals an identical bias towards transitions with a minimal
`number of GRT/CRA transversions (Fig. S2). Indeed, a similar
`propensity towards transitions has been noted in numerous animal
`phylogenies [60]. This consistency in mutational pattern suggests
`that
`the mutagenic processes that cause the accumulation of
`mutations
`in somatic tissue are also responsible for clonal
`population variants arising in the maternal germline.
`In addition to 8-oxo-dG, ROS can also cause a number of other
`mutagenic lesions, including thymine glycol and deamination of
`cytidine and adenosine, all of which can induce transition
`mutations
`[37,61,62]. Our data clearly show an excess of
`transitions relative to transversions, which could be consistent
`with oxidatively induced deamination events becoming fixed as
`
`PLOS Genetics | www.plosgenetics.org
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`6
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`September 2013 | Volume 9 |
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`Issue 9 | e1003794
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`00006
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`is well established that ROS production and
`It
`mutations.
`oxidative damage increase with age [17,18,19,20]. Yet, if oxidative
`damage were the main driver for deamination events, this model
`would predict that the relative proportion of transitions should be
`disproportionately higher in aged individuals, which is not the case
`with our data. While we cannot conclusively rule out a role for
`ROS in inducing transition mutations, the excess of transitions
`could additionally be explained by spontaneous deamination of
`either cytidine or adenosine, especially in single-stranded replica-
`tion intermediates, or base misincorporation events by DNA
`polymerase c during genome replication, which has a known
`propensity for
`transition mutations
`[36,39,40]. Overall,
`the
`absence of key mutagenic signatures of oxidative damage argues
`against ROS being the major driver of mutagenesis in mtDNA in
`normal aging brain.
`Several explanations may account for why we observed few 8-
`oxo-dG associated mutations in mtDNA despite an extensive
`literature showing that 8-oxo-dG levels increase with age. First,
`rapid DNA repair may remove 8-oxo-dG prior to genome
`replication and this repair capacity may increase with age [63],
`thereby keeping 8-oxo-dG mutagenesis to a minimum. Secondly,
`mitochondrial quality control pathways may simply eliminate
`mitochondria with damaged mtDNA. Consistent with this idea is
`the observation that oxidatively damaged mtDNA rapidly
`disappears from cells treated with H2O2 [64]. In addition, the
`cellular levels of parkin, a major component of the quality control
`pathway involved in mitochondrial
`turnover,
`increase under
`conditions of high oxidative stress [65]. Finally, DNA polymerase
`c itself may actively discriminate against incorporating 8-oxo-dG
`[66]. Regardless of the mechanism, our data suggest that cells have
`evolved one or more strategies
`to effectively deal with the
`challenge of replicating mtDNA in the highly damaging environ-
`ment of the mitochondria.
`We find a striking excess of mutations in the D-loop in young
`individuals; however, D-loop mutations accumulate during aging
`at the same rate as other parts of the mitochondrial genome,
`consistent with the D-loop not being inherently error prone.
`Instead, our data suggest that young individuals are born with a
`higher aggregate mutation burden in the D-loop relative to the rest
`of
`the genome,
`thus preserving the D-loop’s disproportionate
`mutation load as mutations accumulate during life. The higher
`p