The multi-tissue landscape of somatic mtDNA mutations indicates tissue-specific accumulation and removal in aging
- Department of Laboratory Medicine and Pathology, University of Washington, United States
- Department of Biochemistry, University of Washington, United States
- Department of Radiology, University of Washington, United States
- Department of Biology, University of Washington, United States
Figures
Figure 1 with 4 supplements
Frequency of somatic mitochondrial genome (mtDNA) mutations increase with age and is tissue-specific.
(A) The frequency by which single-nucleotide variants (SNVs) were detected in all sequenced bases in either young (~5-months of age) or old (26-months of age) tissues arranged from highest to lowest …
Figure 1—figure supplement 1
Mean post-consensus ‘duplex’ depth for young (4.5 months) tissues.
Variant occurring within the masked regions (vertical lines) or positions with less than a post-consusensus depth of 100× were ignored. Gray shading = standard deviation of the mean for N=5 mice.
Figure 1—figure supplement 2
Mean post-consensus ‘duplex’ depth for old (26 months) tissues.
Variant occurring within the masked regions (vertical lines) or positions with less than a post-consusensus depth of 100× were ignored. Gray shading = standard deviation of the mean for N=6 mice.
Figure 1—figure supplement 3
Mitochondrial genome (mtDNA) copy shows no correlation with age, intervention, or mutation frequency.
(A) mtDNA:nDNA ratio varies considerably between the eight tissue types, but does not change with age or treatment with elamipretide (ELAM). (B) Single-nucleotide variant (SNV) mutation frequency …
Figure 1—figure supplement 4
Blood does not significantly contribute to the differences in mutation frequency observed across tissues.
A separate cohort of NIA male aged mice (26 months, N=3) were transcardially perfused with 1× PBS prior to organ harvest. Collected tissues (kidney, liver, hippocampus, cerebellum, and skeletal …
Figure 2
Somatic single-nucleotide variant (SNV) mutations increase linearly with age.
Linear regression of total SNV mutation frequency vs. age in (A) skeletal muscle, (B) brain, and (C) liver. Black = data from Arbeithuber et al.; purple = data from this study; shaded area = 95% …
Figure 3 with 3 supplements
Mutation spectra of somatic mitochondrial genome (mtDNA) mutations demonstrate tissue-specific distribution of mutation types.
(A) Single-nucleotide variant (SNV) frequency by mutation type for young (~5-months of age) tissues shows that replication/deamination-linked G→A/C→T mutations largely dictate overall SNV mutation …
Figure 3—figure supplement 1
Relative proportion of different mutation types varies across tissues.
(A) Young. (B) Old.
Figure 3—figure supplement 2
The single-strand consensus G→T and C→A frequency does not vary across tissues in old mice.
K=kidney; L=liver; RC = retinal pigmented epithelium (RPE)/choroid; R=retina; Hi = hippocampus; C=cerebellum; M=skeletal muscle; He = heart. Significance tested with Welch's t-test. Error bars = …
Figure 3—figure supplement 3
Treatment with FPG does not affect the G→T/C→T mutation frequency.
Skeletal muscle from old skeletal muscle was treated with FPG (New England Biolabs) post adapter ligation but before the first PCR, using the manufacturer’s instructions. Untreated with processed …
Figure 4 with 1 supplement
Clonal expansion of somatic mitochondrial genome (mtDNA) mutations is tissue-specific.
(A) Frequency of mtDNA clones detected in each tissue shows an increase in detection of clones with age in all tissues. Note that y-axes are set for each tissue (N=5 for young; N=6 for old, error = …
Figure 4—figure supplement 1
Mitochondrial genome (mtDNA) variant clones in blood are not a significant contributor to age-related to clonal expansions.
A separate cohort of NIA male aged mice (26 months, N=3) were transcardially perfused with 1× PBS prior to organ harvest. Collected tissues (kidney, liver, hippocampus, cerebellum, and skeletal …
Figure 5
Spectral analysis of clonal somatic mitochondrial genome (mtDNA) mutations suggests removal of reactive oxygen species (ROS)-linked mutations.
(A) Frequency of heteroplasmic clones in young mice shown as clone frequency for each mutation class and tissue. (B) Frequency of heteroplasmic clones in aged mice for each mutation class and …
-
Figure 5—source data 1
Speadsheet containing the calculation of expected vs observed clone counts for Figure 5C.
- https://cdn.elifesciences.org/articles/83395/elife-83395-fig5-data1-v2.xlsx
Figure 6 with 5 supplements
Late-life treatment with mitochondrially targeted interventions reduces somatic mitochondrial genome (mtDNA) mutation burden and is consistent with a mechanism of active reactive oxygen species (ROS)-linked mutation removal.
Mutation spectra show that aged mice treated for 8 weeks with either elamipretide (Elam, diagonal striped bars) or nicotinamide mononucleotide (NMN, horizontal striped bars) have decreased frequency …
Figure 6—figure supplement 1
Mean post-consensus ‘duplex’ depth for aged (26 months) elamipretide-treated tissues.
Variant occurring within the masked regions (vertical lines) or positions with less than a post-consusensus depth of 100× were ignored. Gray shading = standard deviation of the mean for N=5 mice.
Figure 6—figure supplement 2
Mean post-consensus ‘duplex’ depth for aged (26 months) nicotinamide mononucleotide-treated tissues.
Variant occurring within the masked regions (vertical lines) or positions with less than a post-consusensus depth of 100× were ignored. Gray shading = standard deviation of the mean for N=3 mice.
Figure 6—figure supplement 3
Elamipretide (Elam) and nicotinamide mononucleotide (NMN) do not affect the overall mitochondrial genome (mtDNA) mutation frequency.
Overall point mutation frequency for Old (NT), Old+Elam, and Old+NMN separated by tissue. Dunnett’s test with untreated old as the control was used to test for significance. N=6 for Old, N=5 for …
Figure 6—figure supplement 4
Per gene dN/dS ratio shows no apparent selection across age, tissues, and interventions.
Variants for each sample were separated by protein coding gene and the dN/dS ratio calculated using the dNdScv R package using the median depth for each gene as a covariate. dN/dS ratios from the …
Figure 6—figure supplement 5
Late-life treatment with mitochondrially targeted interventions does not reduce somatic mitochondrial genome (mtDNA) mutation burden in retinal pigmented epithelium (RPE)/choroid (pink), retina (cyan), hippocampus (purple), or cerebellum (brown).
One-way ANOVA for each mutation class within tissue with Dunnett’s multiple comparison test compared to untreated aged control group was performed with no significant differences or trends detected. …
Additional files
-
Supplementary file 1
File containing Supplementary Tables 1-5.
Table 1. Sequence of the 96 defined unique molecular identifier (UMI) duplex sequencing adapters. Sequence is provided in 5’→3’ orientation. Complementary UMI sequences are highlighted in red. Table 2. Sequence of the mouse-specific capture probes against the mtDNA. Sequence is provided 5'→3’ orientation. Biotin moiety is denoted by ‘/5Biosg/’. Table 3. Summary of duplex sequencing data. Summary of the samples sequenced, including assay performance metrics, including mitochondrial genome (mtDNA) enrichment specificity, family size, and consensus metrics, bases sequenced, sequencing depth, mutation counts, and mutation frequencies. Table 4. Mitochondrial genome (mtDNA) to nuclear genome (nDNA) copy number ratio data. Summary of the samples used to determine mtDNA and nDNA copy numbers. ND = not determined. Table 5. Genome coordinates of regions masked in the analysis. Coordinates are 1-indexed and columns are in .bed format order.
- https://cdn.elifesciences.org/articles/83395/elife-83395-supp1-v2.xlsx
-
Supplementary file 2
List of all detected mutations.
- https://cdn.elifesciences.org/articles/83395/elife-83395-supp2-v2.zip
-
MDAR checklist
- https://cdn.elifesciences.org/articles/83395/elife-83395-mdarchecklist1-v2.pdf
