Circulating mitochondrial DNA in patients in the ICU as a marker of mortality: derivation and validation

Abstract

Background: Mitochondrial DNA (mtDNA) is a critical activator of inflammation and the innate immune system. However, mtDNA level has not been tested for its role as a biomarker in the intensive care unit (ICU). We hypothesized that circulating cell-free mtDNA levels would be associated with mortality and improve risk prediction in ICU patients.

Methods and findings: Analyses of mtDNA levels were performed on blood samples obtained from two prospective observational cohort studies of ICU patients (the Brigham and Women's Hospital Registry of Critical Illness [BWH RoCI, n = 200] and Molecular Epidemiology of Acute Respiratory Distress Syndrome [ME ARDS, n = 243]). mtDNA levels in plasma were assessed by measuring the copy number of the NADH dehydrogenase 1 gene using quantitative real-time PCR. Medical ICU patients with an elevated mtDNA level (≥3,200 copies/µl plasma) had increased odds of dying within 28 d of ICU admission in both the BWH RoCI (odds ratio [OR] 7.5, 95% CI 3.6-15.8, p = 1×10(-7)) and ME ARDS (OR 8.4, 95% CI 2.9-24.2, p = 9×10(-5)) cohorts, while no evidence for association was noted in non-medical ICU patients. The addition of an elevated mtDNA level improved the net reclassification index (NRI) of 28-d mortality among medical ICU patients when added to clinical models in both the BWH RoCI (NRI 79%, standard error 14%, p<1×10(-4)) and ME ARDS (NRI 55%, standard error 20%, p = 0.007) cohorts. In the BWH RoCI cohort, those with an elevated mtDNA level had an increased risk of death, even in analyses limited to patients with sepsis or acute respiratory distress syndrome. Study limitations include the lack of data elucidating the concise pathological roles of mtDNA in the patients, and the limited numbers of measurements for some of biomarkers.

Conclusions: Increased mtDNA levels are associated with ICU mortality, and inclusion of mtDNA level improves risk prediction in medical ICU patients. Our data suggest that mtDNA could serve as a viable plasma biomarker in medical ICU patients.

Conflict of interest statement

All of the authors except SW declare no conflict of interests. SW has declared: “I've received funding for investigator-initiated studies from Merck, Genzyme, Otsuka, and Satellite Healthcare. I've provided consulting services to BioTrends Research Group, Harvard Clinical Research Institute, and GE Healthcare. I serve on a DSMB for Takeda.”

Figures

Figure 1. Representative standard curve, amplification plot, and dissociation curve, and amplification plot from the patients' samples.

(A) NADH dehydrogenase 1 complementary DNA was serially (1∶10) diluted to prepare a series of calibrators (mtDNA standard) with known DNA copy number. The assay was linear over the range (8.48–848000 copies) of DNA copy numbers (R2 = 0.997866). (B) The amplification plot (ΔRn versus cycle [log] view) shows no irregular amplification for the standard diluents (848,000, 84,800, 8,480, 848, 84.8, 8.48). (C) The dissociation curve shows a single melting temperature of the specific products generated with standard template. Also, there is no melting temperature observed in the no-template control wells (blank). These dissociation curves indicate that the reactions are free of primer-dimer or any other spurious products. (D) Similar amplification plots were observed after qPCR using patients' samples. The amplification curves generated from human samples were paralleled with the curves from standards and were in the range of amplification plots for standard diluents.

Figure 2. Cell-free mtDNA level in the plasma of ICU patients.

Boxplots comparing measures of cell-free mtDNA level (mtDNA [NADH dehydrogenase 1] expressed as copy number/µl of plasma) in the plasma of (A) patients who died within 28 d after ICU admission (red) versus those who survived (gray), (B) patients with (green) versus without (gray) sepsis, and (C) patients with (blue) versus without (gray) ARDS in the BWH RoCI (left) and ME ARDS (right) cohorts. mtDNA copy number in the plasma is presented as median value (black line), interquartile range (box), and 5th and 95th percentiles (whiskers). p-Values are noted in the figure for each cohort.

Figure 3. Survival of BWH RoCI MICU patients stratified by mtDNA level.

Kaplan-Meier estimates of survival for patients who had plasma mtDNA levels ≥3,200 copies/µl (n = 101) and <3,200 copies/µl (n = 99) in the BWH MICU. The middle black line indicates the Kaplan-Meier survival curve for patients who had mtDNA <3,200 copies/µl, with 95% confidence intervals (outer black lines and gray shading). The middle red line indicates the Kaplan-Meier survival curve for patients who had mtDNA ≥3,200 copies/µl, with 95% confidence intervals (outer red lines and pink shading). Survival in patients with mtDNA ≥3,200 copies/µl was significantly lower than in patients with mtDNA <3,200 copies/µl (p-value noted in the figure).

Figure 4. Receiver operating characteristic curves, mtDNA, and death in ICU patients.

Comparisons of receiver operating characteristic (ROC) curves for a clinical model (including age, gender, race/ethnicity, APACHE II score, and sepsis) (solid lines) and a clinical model with an mtDNA level ≥3,200 copies/µl (dashed lines) to predict 28-d mortality in ICUs. (A) The area under the curve was 0.76 for a clinical model and was 0.83 for a clinical model with an mtDNA level ≥3,200 copies/µl in the BWH RoCI cohort. (B) The area under the curve was 0.85 for a clinical model and was 0.86 for a clinical model with an mtDNA level ≥3,200 copies/µl in the ME ARDS cohort when all patients were included. (C) The area under the curve was 0.87 for a clinical model and was 0.89 for a clinical model with an mtDNA level ≥3,200 copies/µl in the subpopulation of ME ARDS MICU patients.