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. 2022 Oct;12(10):e1055.
doi: 10.1002/ctm2.1055.

Novel attributes of cell-free plasma mitochondrial DNA in traumatic injury

Grant T Daly  1 , Viktor M Pastukh  1 , Yong B Tan  2 , C Michael Francis  3 , C Zack Aggen  1 , S Chris Groark  1 , Carson Edwards  1 , Madhuri S Mulekar  4 , Mohammad Hamo  1 , Jon D Simmons  2 , Matthew E Kutcher  5 , Emily M Hartsell  1 , Darrell L Dinwiddie  6 , Zachary M Turpin  7 , Hank W Bass  7 , Justin T Roberts  1 , Mark N Gillespie  1 , Raymond J Langley  1
Affiliations

Novel attributes of cell-free plasma mitochondrial DNA in traumatic injury

Grant T Daly et al. Clin Transl Med. 2022 Oct.
No abstract available

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
RNA target bait‐capture and bioinformatics protocol and nuclear mitochondrial (NUMT) identification. (A) DNA is isolated from plasma or tissue. In the figure, nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) are denoted by colour. DNA isolation and library preparation are applied to all sample DNA, regardless of nuclear or mitochondrial origin. A target bait‐capture kit consists of biotinylated RNA probes complementary to the mitochondrial genome. The probes efficiently bind mtDNA but can also bind homologous NUMT, as illustrated by the DNA fragment half coloured as mitochondrial and half coloured as nuclear. Once enriched, samples are pooled and sequenced on a standard Illumina instrument. From there, a Workflow Description Language pipeline aligns the reads to the whole genome –nuclear and mitochondrial. Three custom C/C++ programs built on the htslib library then call mitochondrial and nuclear coverage, insert (the size of a fragment after end repair and sequencing adapter ligation), and variant calling. (B) Schematic depiction of how target‐bait capture also leads to the sequencing of flanking regions of polymorphic NUMTs. (C) Integrated Genome Viewer (IGV) histograms depicting a specific polymorphic NUMT in a nontransfused patient whereas the second patient lacks this insertion at either t0 or t72 post‐admittance. Subfigures (A) and (B) were prepared in Inkscape. (C) Prepared from IGV.
FIGURE 2
FIGURE 2
Enrichment, mean genome‐wide coverage, fragment length and total variants in trauma patients. (A) Target‐bait capture enrichment in combination with the judicious exclusion of nuclear mitochondrial (NUMT) leads to significant enrichment of the mitochondrial DNA (mtDNA) genome versus whole genome sequencing of cell‐free plasma. Enrichment efficiency was calculated for mean coverage of the mtDNA genome (reads/base) after whole genome sequencing and after target‐bait enrichment in four individual patients. Mean ± standard error of the mean; *p ≤ .05. (B) Normalized mean coverage of mtDNA (mean coverage/mean NUMT coverage), fragment length (mean length, measured in bp) and a number of heteroplasmies for all 30 trauma patients. Hundred base pair nonoverlapping bins of (C). normalized mean coverage across the mtDNA genome and (D) mean fragment length across the ≈16.5 kb expanse of the mitochondrial genome. (E) Heteroplasmic variants in all 30 patients as reported in 100 bp bins, where colour demarcates the variant allele fraction (VAF) of the variants. See “methods” for details.
FIGURE 3
FIGURE 3
Fragment length but not normalized coverage correlate with injury scores or severity. (A–D) Normalized coverage determined by mean coverage/mean nuclear mitochondrial (NUMT) coverage. Systemic inflammatory response syndrome (SIRS) defined by a patient with two or more criteria for systemic inflammatory response syndrome. Acute kidney injury (AKI) was determined by the KDIGO Criterial and while acute lung injury (ALI) was assessed by a PaO2:FiO2 ratio between 100–200 in patients requiring at least 40% FiO2 at 72 h post‐admittance. (E–H) Insert size determined by the mean length of the mitochondrial DNA (mtDNA) fragment (bp) that was sequenced in each patient. Non‐parametric Mann‐Whitney U‐tests were performed to calculate the indicated p‐values for (A–H).
FIGURE 4
FIGURE 4
Solar Manhattan plots depicting plasma mitochondrial DNA (mtDNA) variants associated with severe outcomes. Variants meeting the minimum variant allele fraction density ≥25%; mtDNA region is denoted by coloured sequences. Variants significantly increased for risk of indicated complications located at or outside the red ring. Functional regions of the mitochondrial genome are displayed in the interior, and variants are coloured and shaped by in silico predicted effect. Acute kidney injury (AKI) was associated with multiple significant variants in the D‐loop control region while acute lung injury (ALI)‐related variants were present in D‐loop and cytochrome B regions. Odds ratios for individual variants were calculated. Associations are considered significant at p < .05 and are plotted as the negative log10 of the p‐values at each mitochondrial position (denoted in the figure by any point outside of the red circle). The figure was rendered with the ggbio R package.
See this image and copyright information in PMC

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