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. 2020 Sep 17;21(1):248.
doi: 10.1186/s13059-020-02138-5.

Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging

Scott A Lujan  1 , Matthew J Longley  2 , Margaret H Humble  2 , Christopher A Lavender  3 , Adam Burkholder  3 , Emma L Blakely  4   5 , Charlotte L Alston  4   5 , Grainne S Gorman  4 , Doug M Turnbull  4 , Robert McFarland  4 , Robert W Taylor  4   5 , Thomas A Kunkel  1 , William C Copeland  6
Affiliations

Ultrasensitive deletion detection links mitochondrial DNA replication, disease, and aging

Scott A Lujan et al. Genome Biol. .

Abstract

Background: Acquired human mitochondrial genome (mtDNA) deletions are symptoms and drivers of focal mitochondrial respiratory deficiency, a pathological hallmark of aging and late-onset mitochondrial disease.

Results: To decipher connections between these processes, we create LostArc, an ultrasensitive method for quantifying deletions in circular mtDNA molecules. LostArc reveals 35 million deletions (~ 470,000 unique spans) in skeletal muscle from 22 individuals with and 19 individuals without pathogenic variants in POLG. This nuclear gene encodes the catalytic subunit of replicative mitochondrial DNA polymerase γ. Ablation, the deleted mtDNA fraction, suffices to explain skeletal muscle phenotypes of aging and POLG-derived disease. Unsupervised bioinformatic analyses reveal distinct age- and disease-correlated deletion patterns.

Conclusions: These patterns implicate replication by DNA polymerase γ as the deletion driver and suggest little purifying selection against mtDNA deletions by mitophagy in postmitotic muscle fibers. Observed deletion patterns are best modeled as mtDNA deletions initiated by replication fork stalling during strand displacement mtDNA synthesis.

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Conflict of interest statement

The authors claim no competing interests. The views expressed in this publication are those of the author(s) and not necessarily those of the NIH, NIEHS, NHS, the National Institute for Health Research, or the Department of Health and Social Care.

Figures

Fig. 1
Fig. 1
The human mtDNA reference map and the mtDNA deletion mapping pipeline. a Human mtDNA reference map, color-coded by feature. Inset: respiratory chain complexes encoded by mtDNA (color) and nuclear genes (gray). MtDNA-encoded genes and feature abbreviations: tRNA genes, IUPAC single-letter amino acid codes; rRNA genes, sedimentation coefficients (e.g., 16S); previously proposed heavy and light strand features, _H and _L; promoters, P_; replication origins, O_ (e.g., PH1, OL); and MT- prefixes are omitted. The 7S-3′-terminus and oriL are indicated (red and green arrows). b LostArc method outline. c The mtDNA fractions (mean ± 95% CL) indicate enrichment during library preparation and a subsequent lack of selection in sequencing (qPCR for steps 1, 3, and 4b, fraction of reads mapped to mtDNA reference for step 7). See also Additional file 1: Fig. S1. d Estimated mtDNA frequencies. Species with frequencies over 5 × 10−4 must reside in multiple truncated fibers (i.e., they predate adulthood or mark mutational hotspots). Deletions observed ≥ 2x must have ≥ 20 copies in the muscle sample. Deletions observed once will have frequencies inflated to roughly 1/depth. These collectively represent all less frequent species
Fig. 2
Fig. 2
Deletion frequencies and ablation levels of mtDNA vary with age, disease, and mapped location. a–e Gwt muscle samples are represented by triangles, and Gvar samples are represented by circles. Among wild type samples, one young and one aged sample are highlighted (M03, biopsied at 23 years, in black and M17, biopsied at 83 years, in red, respectively). a Among Gwt muscle samples, deletion loads are independent of age (per Mbp mapped to mtDNA reference). b The fraction of mtDNA remaining for two Gwt and two Gvar samples. A linearized mtDNA map (see Fig. 1a) is shown above the graph with key features indicated: oriL (green); 7S-DNA 3′-end (red); minor arc (magenta); and major arc (teal). c Ablation (area above the curve in b) depends upon age, as seen in Gwt samples. d Ablation at biopsy is higher and more varied among Gvar samples. Ablation increases with age between well-spaced Gvar samples: A467T;A467T (dark blue); and heterozygous Y955C (purple). e The minimum estimated ablation at onset in Gvar samples exceeds the maximum observed ablation at biopsy in Gwt samples, implying a threshold for clinical symptoms (dashed line). f A deletion length spectrum example (1 bp bins; Gvar sample M24). Very large deletions (> 15 kbp) may indicate insertions, primarily within the control region. For all samples, see LostArc Reports in Supplemental Information (also see Additional file 1: Figs. S4 and S5). Major and minor arc sizes are shown below the histogram (teal and magenta). g Deletion length spectra (400 bp bins) for Gwt and variant (A467T;A467T) samples of similar ages
Fig. 3
Fig. 3
Size, position, frequency, and context of mtDNA deletions. a Frequencies of ≥ 20 bp deletions by Gwt age cohort and colored by degree of terminal microhomology (TMH; red to blue for 0 to 9 bp; green for ≥ 10 bp). TMH refers to identical sequences found in one deletion terminus and in the region flanking the other terminus. For simplicity, error bars (standard error) are only shown for 0, 5, and ≥ 10 bp of TMH. b–d Example visualizations of deletion frequencies by subsets of size, position, frequency, and sequence context (Gvar sample M24) with versions of the mtDNA map (b linear; c, d circular). The 7S-3′ terminus and oriL are indicated by red and green arrows. b A Bubble Map of deletions by terminal positions. Termini are classified starting from the center of a deletion (see inset diagram): one terminus lies in the light strand 5′-direction (red) and the other lies in the heavy strand 5′-direction (blue). Bubble area scaled by deletion frequency and colored by surrounding repeat tract (gray) or by the length of TMH: 0 bp (red); 1–9 bp (blue); ≥ 10 bp (green). The diagonal contains deletions of under 100 bp (peak in Fig. 2f). Minor and major arc deletions fall in the magenta and cyan areas (broad overlaid ranges of peaks in Fig. 2f). Plots equivalent to b may be found for all samples in Additional file 2: LostArc Reports (also see Additional file 1: Figs. S4 and S5). c An Arc Map with data as in b. Line width and transparency code for frequency (see deletion frequency key). Colors code for deletion size and terminal homology (see Length/homology key). Note the paucity of extensive TMH. The thin green arc from positions 8471 to 13,447 has been termed the common deletion. d As per c, but colors code for the degree of deletion terminus clustering (see Terminal Cluster key). Plots equivalent to c, d may be found for Gvar samples in Supplemental LostArc Arc Maps
Fig. 4
Fig. 4
Unsupervised exploratory analysis of mtDNA deletion patterns. Hierarchical clustering revealed similarities between samples and principal component analysis (PCA) revealed the underlying patterns that explain the most variation between samples. Both analyses used the fraction of deletions, binned by terminus positions (80 × 80 bins), rather than deletion frequencies or mtDNA ablation levels. Deletions of < 40 bp and deletions mapped to the T-shaped white region (b, c) were excluded as possible false positives (see Methods). a Hierarchical clustering by Euclidean distance separates samples into three clades. Gwt samples form a WT clade and sort largely by age at biopsy (black to red; see Additional file 1: Table S1). Gvar samples are “paraphyletic,” i.e., some have patterns that are more like Gwts (AT-WS clade) than like other Gvars (TI-PL clade). Colored squares below the dendrogram indicate POLG genotypes, with ablation levels shown below. Similar Gvar genotypes tend to cluster. Purple bars below the dendrogram illustrate ablation levels. b, c A representative subset of Gvar samples was used for PCA (see the “Materials and methods” section): all Gwts; AT-WS and TI-PL clade representatives; and cladeless sample M24. b The 1st PCA component vector (deletion terminus positions as per Fig. 3b) colored by PCA weight (red to blue for ≥ 0.04 to ≤ − 0.04). c The 3rd PCA component weight vector. d–g Samples were scored using PCA weights. Black curves are parabolas fit to Gwt data (R2 inset). d The 1st component distinguishes Gwt (triangles) from Gvar deletion patterns, except M24 (orange diamond with black border). e 3rd component scores correlate with age at biopsy, regardless of POLG disease state. f These two components separate Gwt and Gvar clusters by deletion pattern (naïve to disease, age, and overall deletion frequency). g The 2nd PCA component was spent describing unique features of the disease state M24. In order to display minor arc features, the vectors in b, c were recalculated without M24
Fig. 5
Fig. 5
Deletion maps support a strand displacement mode of mtDNA replication. Deletion patterns were compared to four replication modes. a–d Heavy and light strands are shown in red and blue, with template strands darker. Arrows indicate 5′-to-3′ synthesis directions. Continuously and discontinuously replicated strands are shown as solid and dashed, respectively. Potential heavy and light strand replication origins (labeled oH and oL) are indicated by triangles (gray when unused). See the “Results” section for detailed descriptions of the a strand displacement mode, b the rolling circle mode proposed for Caenorhabditis elegans mtDNA, c a unidirectional, strand-synchronous mode as proposed for Drosophila mtDNA and older RITOLS and Bootlace modes, and d strand-coupled DNA replication. e Deletion arcs, scaled by individual deletion frequency and colored by terminus frequency in 100 bp bins, cluster near oriL and the 3′-terminus of 7S-DNA. See Supplemental Arc Plots for all Gvar samples. f–h Heavy- and light-strand-5′-terminus frequencies (lighter blue and red; 250 bp bins; excluding deletions with TMH ≥ 10 bp) reveal double-bowtie patterns in Gvar and aged Gwt samples (see 1st PCA component in Fig. 4b). Except in Gwt samples under 65 years of age, these patterns fit replication/deletion Monte Carlo models (darker curves; simulation 6 in Additional file 1: Fig. S6a). f Gvar example M38. g Younger Gwt example M03. h Older Gwt example M17. i Mean deletion lengths, from Monte Carlo regression, increased with age in Gwt (triangles) and Gvar (circles) samples
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