Cell-free Mitochondrial DNA Research Articles

Plasma levels of mitochondrial and nuclear DNA in patients with massive pulmonary embolism in the emergency department: a prospective cohort study

IntroductionCell-free plasma mitochondrial DNA (mt-DNA) and nuclear DNA (n-DNA) are biomarkers with prognostic utility in conditions associated with a high rate of cell death. This exploratory study aimed to determine the plasma levels of both nucleic acids in patients with massive and submassive pulmonary embolism (PE) and to compare them with other biomarkers, such as heart-type fatty acid-binding protein (H-FABP) and troponin I (Tn-I)MethodsThis was a prospective observational study of 37 consecutive patients with massive PE, 37 patients with submassive PE, and 37 healthy subjects. Quantifications of plasma mt-DNA and n-DNA with real-time quantitative polymerase chain reaction (PCR), and plasma H-FABP and Tn-I by commercial assays, were done on blood samples drawn within 4 hours after presentation at the emergency department.ResultsPlasma mt-DNA and n-DNA concentrations were much higher in patients with massive PE (median, 2,970 GE/ml; interquartile range (IQR), 1,050 to 5,485; and 3,325 GE/ml, IQR: 1,080 to 5,790, respectively) than in patients with submassive PE (870 GE/ml and 1,245 GE/ml, respectively; P < 0.01) or controls (185 GE/ml and 520 GE/ml, respectively). Eighteen patients with massive PE died of a PE-related cause by day 15 of observation. Plasma mt-DNA and n-DNA values were 2.3-fold and 1.9-fold higher in the subgroup of nonsurviving patients than in survivors. H-FABP and Tn-I values were also higher in patients with massive PE who died (7.3 ng/ml and 0.023 ng/ml, respectively) than in those who survived (6.4 ng/ml, and 0.016 ng/ml, respectively). By receiver operating curve (ROC) analysis, the best cutoff values for predicting 15-day mortality were 3,380 GE/ml for mt-DNA, 6.8 ng/ml for H-FABP, 3,625 GE/ml for n-DNA, and 0.020 ng/ml for Tn-I, based on the calculated areas under the curve (AUCs) of 0.89 (95% confidence interval (CI), 0.78 to 0.99), 0.76 (95% CI, 0.69 to 093), 0.73 (95% CI, 0.58 to 0.91), and 0.59 (95% CI, 0.41 to 0.79), respectively. By stepwise logistic regression, a plasma mt-DNA concentration greater than 3,380 GE/ml (adjusted odds ratio (OR), 8.22; 95% CI, 1.72 to 39.18; P < 0.001) and a plasma value of H-FBAP >6.8 ng/ml (OR, 5.36; 95% CI, 1.06 to 27.08; P < 0.01) were the only independent predictors of mortality.Conclusionsmt-DNA and H-FBAP might be promising markers for predicting 15-day mortality in massive PE, with mt-DNA having better prognostic accuracy.

Cell-free DNA as a promising marker for risk stratification of pulmonary embolism

Arnalich and colleagues reported an interesting clinical study that evaluated cell-free plasma mitochondrial and nuclear DNA concentrations in patients with pulmonary embolism (PE) [1]. They found increased circulating mitochondrial and nuclear DNA levels, as well as alterations in other cardiac biomarkers (heart-type fatty acidbinding protein and troponin I) in patients with massive PE compared with those found in patients with submassive PE [1]. Importantly, all these biomarkers were found at higher concentrations in fatal massive PE than in nonfatal PE [1]. I would like to add experimental information associating cell-free DNA and acute pulmonary thromboembolism (APT). Experimental evidence showed that cell-free DNA concentrations increase in proportion to the severity of experimental APT [2]. In addition, these previous findings suggest that lysed cells from the thrombi are

Mutations in circulating mitochondrial DNA: Cassandra of oral cancer?

Cell-free circulating nucleic acids in human blood are increasing being researched as a source of diagnostic and prognostic biomarkers for clinical oncology. High copy number per cell and frequent mutations in various malignancies make mitochondrial genome an attractive target for such an investigation, but practical development and validation of biomarkers based on cell-free mitochondrial DNA has been lagging. Uzawa and colleagues report in the July issue of Oncotarget that in a retrospective study of patients with oral cancer the load of mutant mitochondrial DNA in patient's serum was a strong indicator of postoperative recurrence. Based on these observations, the predictive value of circulating mutant mitochondrial DNA merits further evaluation in patients with oral and other malignancies.

Cell-free Circulating Mitochondrial DNA in the Serum: A Potential Non-invasive Biomarker for Ewing's Sarcoma

Altered levels of circulating cell-free mitochondrial DNA (ccf mtDNA) have been recently detected in several cancer types and have been proposed as a promising noninvasive diagnostic or prognostic biomarker. There has been no report regarding quantification of ccf mtDNA in patients with Ewing's sarcoma (EWS). Ccf mtDNA copy number in serum samples obtained from 25 patients with EWS as well as 20 age-matched individuals were detected by quantitative real-time PCR assays. A receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic applicability of serum ccf mtDNA as a noninvasive biomarker for discriminating between patients and healthy cohorts. The potential connection between ccf mtDNA levels and various clinicopathological factors of EWS was also determined. We found that levels of ccf mtDNA in the serum of EWS patients were significantly lower than in healthy controls. The receiver operating curve analysis demonstrated that using serum ccf mtDNA content as a molecular marker allowed distinguishing between EWS patients and healthy subjects with a sensitivity of 76.1 and a specificity of 68.4%. In addition, serum levels of ccf mtDNA were associated with the status of tumor metastasis. These results suggest that serum ccf mtDNA has the potential capacity as a novel and convenient noninvasive biomarker for molecular diagnosis of EWS. Scrutinizing quantitative changes of serum ccf mtDNA may provide valuable clues for better management of EWS patients.

Strategies of reducing input sample volume for extracting circulating cell-free nuclear DNA and mitochondrial DNA in plasma

Circulating cell-free (ccf) DNA in blood has been suggested as a potential biomarker in many conditions regarding early diagnosis and prognosis. However, misdiagnosis can result due to the limited DNA resources in Biobank's plasma samples or insufficient DNA targets from a predominant DNA background in genetic tests. This study explored several strategies for an efficient DNA extraction to increase DNA amount from limited plasma input. Ccf plasma DNA was extracted with three different methods, a phenol-chloroform-isoamylalcohol (PCI) method, a High Pure PCR Template Preparation Kit method and a method used for single cell PCR in this group. Subsequently, the total DNA was measured by Nanodrop and the genome equivalents (GE) of the GAPDH housekeeping gene and MTATP 8 gene were measured using a multiplex real-time quantitative PCR for the quantitative assessment of nDNA and mtDNA. Instead of 400-800 μL (routine input in the laboratory), 50 μLof plasma input enabled the extraction of ccf DNA sufficient for quantitative analysis. Using the PCI method and the kit method, both nDNA and mtDNA could be successfully detected in plasma samples, but nDNA extracted using protocol for single cell PCR was not detectable in 25% of plasma samples. In comparison to the other two methods, the PCI method showed lower DNA purity, but higher concentrations and more GE of nDNA and mtDNA. The PCI method was more efficient than the other two methods in the extraction of ccf DNA in plasma. Limited plasma is available for ccf DNA analysis.

Circulating mitochondrial DNA in the serum of patients with testicular germ cell cancer as a novel noninvasive diagnostic biomarker

To analyse the diagnostic and prognostic value of cell-free mitochondrial (mt)DNA in patients with testicular cancer, as increased levels of cell-free circulating mtDNA have been reported in patients with cancer. In all, 74 patients with testicular cancer (seminoma in 39, nonseminoma in 35) and 35 healthy individuals were included in the study. Circulating DNA was isolated from 1 mL of serum. A quantitative real-time polymerase chain reaction was used to analyse the levels of a 79-bp (mtDNA-79) and 220 bp (mtDNA-220) fragment of the mitochondrial specific 16S-RNA. The mtDNA integrity was expressed as the ratio of mtDNA-220 to mtDNA-79. mtDNA-79 and mtDNA-220 levels were significantly (P < 0.001) greater in patients with testicular cancer than in healthy individuals. The mtDNA integrity was similar in patients and healthy controls (P = 0.435). Receiver operator characteristic curve analysis showed that cell-free mtDNA (mtDNA-79) levels distinguished, with a sensitivity of 59.5% and a specificity of 94.3%, between patients and healthy individuals (area under curve, 0.787). Also, mtDNA-79 levels could be used to distinguish between patients (31) with conventional markers (alpha-fetoprotein, human chorionic gonadotrophin, placental alkaline phosphatase and lactate dehydrogenase) within normal ranges and healthy individuals, with a sensitivity of 64.5% and specificity of 91.4% (area under curve 0.797). Cell-free mtDNA levels were not correlated with any clinicopathological variable (pT stage, lymph node invasion, vascular invasion, clinical stage, International Germ Cell Cancer Collaborative Group classification, tumour markers; all P > 0.05). Cell-free mtDNA levels are greater in patients with testicular cancer and might provide valuable information for managing patients with testicular anomalies, especially those with normal levels of established tumour markers.

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.

Levels of Circulating Cell-Free Nuclear and Mitochondrial DNA in Benign and Malignant Ovarian Tumors

To analyze the levels of circulating cell-free nuclear DNA and circulating cell-free mitochondrial DNA in patients with benign and malignant ovarian tumors using a gold-standard assay and to investigate whether quantitative alterations of the circulating cell-free species have values in the management of the patients. One hundred four patients were recruited for this study. We developed a quantitative, multiplex polymerase chain reaction to measure the levels of circulating cell-free nuclear DNA and circulating cell-free mitochondrial DNA in serum and plasma of patients with epithelial ovarian cancer, benign epithelial ovarian tumors, or endometriosis. The levels of the circulating cell-free DNA were compared with those of a healthy, age-matched control group. The patients with epithelial ovarian cancer had significantly higher amounts of circulating cell-free nuclear DNA and circulating cell-free mitochondrial DNA in plasma compared with the healthy control group (mean of nuclear DNA 10,723/2,591 and mean of mitochondrial DNA 4,918,978/2,294,264, P=.009 and 0.022, respectively) and with the other group with benign ovarian diseases (mean of nuclear DNA 10,723/2,965 and mean of mitochondrial DNA 4,918,978/1,597,551, P=.027 and 0.002, respectively). However, no relationship between levels of the circulating cell-free DNA and the pathological parameters as well as CA 125 measurement in patients with epithelial ovarian cancer was found. A significant difference between the epithelial ovarian cancer and endometriosis group was found in circulating cell-free mitochondrial DNA but not in circulating cell-free nuclear DNA (mean of mitochondrial DNA 4,918,978/2,273,988 and mean of nuclear DNA 10,723/3,291, P=.013 and 0.105, respectively). Elevated levels of circulating cell-free nuclear DNA and circulating cell-free mitochondrial DNA in epithelial ovarian cancer may have diagnostic value. Our finding suggests that the circulating molecules might be potential biomarkers in the disease.

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Mechanisms of Endothelial Cell Heterogeneity in Health and Disease

See related article, pages 200–208 The endothelium is an expansive spatially distributed organ.1 Endothelial cells participate in a large number of physiological processes including the control of vasomotor tone, the trafficking of cells and nutrients, the regulation of permeability, and the maintenance of blood fluidity. In addition, the endothelium mediates new blood vessel formation, contributes to the balance of pro- and antiinflammatory mediators, and may play a role in antigen presentation. In accomplishing these tasks, the endothelium exhibits a remarkable “division of labor”. For example, arteriolar endothelium is primarily responsible for mediating vasomotor tone; endothelium in postcapillary venules regulates leukocyte trafficking; capillary endothelial cells display organ-specific barrier properties (eg, blood brain barrier versus fenestrated, discontinuous endothelium in hepatic sinusoids); and endothelial cells from different vascular beds balance local hemostasis via the expression of site-specific patterns of anticoagulants and procoagulants.2 In recent years, in vivo phage display and direct proteome mapping of the intact vasculature have revealed a rich diversity in endothelial cell surface markers.3,4 Any consideration of the mechanisms underlying endothelial heterogeneity is best framed around the time-honored debate of nature versus nurture (which will be addressed here in reverse order) (Figure). Mechanisms of endothelial cell heterogeneity. Relative importance of epigenetics and microenvironment in mediating site-specific phenotypes is indicated by +. The table is designed to provide a conceptual framework; the scores are largely speculative and will require ongoing experimental validation. ### Nurture Site-specific endothelial cell phenotypes may be initiated and maintained by signals residing in the extracellular environment. The endothelium is analogous to a barcode reader, constantly taking inventory of its surrounding extracellular environment on the luminal side (circulating blood and its constituents), the abluminal side, and at the endothelial junctions. Environmental cues may be classified into biomechanical or biochemical. Biochemical forces include shear stress and strain. …

Presence of mitochondrial tRNA Leu(UUR) A to G 3243 mutation in DNA extracted from serum and plasma of patients with type 2 diabetes mellitus

Aims/Background—An A to G substitution at base pair 3243 in the mitochondrial tRNA Leu(UUR) gene (mt3243) is commonly associated with maternally inherited diabetes and deafness, and other diseases. It is...