RESUMO
Cardiovascular disease (CVD) is a leading global cause of mortality, difficult to predict in advance. Evidence indicates that the copy number of mitochondrial DNA (mtDNAcn) in blood is altered in individuals with CVD. MtDNA released into circulation may act as a mediator of inflammation, a recognized factor in the development of CVD, in the long distance. This pilot study aims to test if levels of mtDNAcn in buffy coat DNA (BC-mtDNA), in circulating cellfree DNA (cf-mtDNA), or in DNA extracted from plasma extracellular vesicles (EV-mtDNA) are altered in CVD patients and if they can predict heart attack in advance. A group of 144 people with different CVD statuses (50 that had CVD, 94 healthy) was selected from the LifeLines Biobank according to the incidence of new cardiovascular event monitored in 6 years (50 among controls had heart attack after the basal assessment). MtDNAcn was quantified in total cf-DNA and EV-DNA from plasma as well as in buffy coat. EVs have been characterized by their size, polydispersity index, count rate, and zeta potential, by Dynamic Light Scattering. BC-mtDNAcn and cf-mtDNAcn were not different between CVD patients and healthy subjects. EVs carried higher mtDNAcn in subject with a previous history of CVD than controls, also adjusting the analysis for the EVs derived count rate. Despite mtDNAcn was not able to predict CVD in advance, the detection of increased EV-mtDNAcn in CVD patients in this pilot study suggests the need for further investigations to determine its pathophysiological role in inflammation.
Assuntos
Doenças Cardiovasculares , Ácidos Nucleicos Livres , Variações do Número de Cópias de DNA , DNA Mitocondrial , Vesículas Extracelulares , Humanos , DNA Mitocondrial/genética , DNA Mitocondrial/sangue , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Masculino , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Feminino , Projetos Piloto , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/sangue , Pessoa de Meia-Idade , Estudos de Casos e Controles , Idoso , Estudos ProspectivosRESUMO
Novel screening techniques for early detection of lung cancer are urgently needed. Profiling circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping. However, both the scarcity and short half-life of ctDNA substantially limit the sensitivity and clinical utility of ctDNA detection methodologies. Our discovery that red blood cells (RBCs) sequester mitochondrial DNA opens a new avenue for detecting circulating nucleic acids, as RBCs represent an unrecognized reservoir of circulating nucleic acid. Here, we show that RBCs acquire tumor DNA following coculture with lung cancer cell lines harboring Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations. RBC-bound tumor DNA is detectable in patients with early-stage non-small cell lung cancer (NSCLC) but not in healthy controls by qPCR. Our results collectively uncover a previously unrecognized yet easily accessible reservoir of tumor DNA, offering a promising foundation for future RBC-based tumor diagnostics.NEW & NOTEWORTHY We present a novel method for lung cancer detection by revealing RBCs as a reservoir for tumor DNA, overcoming the limitations of current circulating tumor ctDNA methodologies. By demonstrating that RBCs can capture tumor DNA, including critical mutations found in lung cancer, we provide a promising, biopsy-free avenue for early cancer diagnostics. This discovery opens up exciting possibilities for developing RBC-based diagnostic tools, significantly enhancing the sensitivity and clinical utility of noninvasive cancer detection.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , DNA Tumoral Circulante , Eritrócitos , Neoplasias Pulmonares , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/diagnóstico , Eritrócitos/metabolismo , DNA Tumoral Circulante/genética , DNA Tumoral Circulante/sangue , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Mutação , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/sangue , Proteínas Proto-Oncogênicas p21(ras)/genética , Masculino , Feminino , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , DNA de Neoplasias/sangue , DNA de Neoplasias/genéticaRESUMO
Mitochondrial DNA copy number (mtDNA-CN) is a proxy for mitochondrial function and is associated with aging-related diseases. However, it is unclear how mtDNA-CN measured in blood can reflect diseases that primarily manifest in other tissues. Using the Genotype-Tissue Expression Project, we interrogated relationships between mtDNA-CN measured in whole blood and gene expression from whole blood and 47 additional tissues in 419 individuals. mtDNA-CN was significantly associated with expression of 700 genes in whole blood, including nuclear genes required for mtDNA replication. Significant enrichment was observed for splicing and ubiquitin-mediated proteolysis pathways, as well as target genes for the mitochondrial transcription factor NRF1. In nonblood tissues, there were more significantly associated genes than expected in 30 tissues, suggesting that global gene expression in those tissues is correlated with blood-derived mtDNA-CN. Neurodegenerative disease pathways were significantly associated in multiple tissues, and in an independent data set, the UK Biobank, we observed that higher mtDNA-CN was significantly associated with lower rates of both prevalent (OR = 0.89, CI = 0.83; 0.96) and incident neurodegenerative disease (HR = 0.95, 95% CI = 0.91;0.98). The observation that mtDNA-CN measured in blood is associated with gene expression in other tissues suggests that blood-derived mtDNA-CN can reflect metabolic health across multiple tissues. Identification of key pathways including splicing, RNA binding, and catalysis reinforces the importance of mitochondria in maintaining cellular homeostasis. Finally, validation of the role of mtDNA CN in neurodegenerative disease in a large independent cohort study solidifies the link between blood-derived mtDNA-CN, altered gene expression in multiple tissues, and aging-related disease.
Assuntos
Variações do Número de Cópias de DNA , DNA Mitocondrial/sangue , DNA Mitocondrial/genética , Expressão Gênica , Doenças Neurodegenerativas/genética , Estudos de Coortes , Feminino , Humanos , Masculino , Especificidade de Órgãos/genéticaRESUMO
BACKGROUND AND OBJECTIVES: Platelet storage lesion (PSL) adversely affects the quality of platelet concentrates (PCs). Platelets are prone to activation during storage. Moreover, elevated free mitochondrial DNA (mtDNA) levels in PCs are associated with a higher risk of adverse transfusion reactions. Therefore, we aimed to evaluate the correlation between platelet activation markers and mtDNA release during PC storage. MATERIALS AND METHODS: Six PCs prepared by the platelet-rich plasma method were assessed for free mtDNA copy number using quantitative real-time PCR and CD62P (P-selectin) expression by flow cytometry on days 0 (PC collection day), 3, 5 and 7 of storage. Lactate dehydrogenase (LDH) activity, pH, platelet count, mean platelet volume (MPV) and platelet distribution width (PDW) were measured as well. The correlation between free mtDNA and other PSL parameters, and the correlation between all parameters, was determined. RESULTS: Significant increases in free mtDNA, MPV and PDW, and a significant decrease in platelet count and pH were observed. CD62P expression and LDH activity elevated significantly, particularly on storage days 5-7 and 0-3, respectively. Moreover, a moderate positive correlation (r = 0.61) was observed between free mtDNA and CD62P expression. The r values between free mtDNA and LDH, pH, platelet count, MPV and PDW were 0.81, -0.72, -0.49, 0.81 and 0.77, respectively. CONCLUSION: The interplay between platelet activation and mtDNA release in promoting PSL in PCs may serve as a promising target for future research on applying additive solutions and evaluating the quality of PCs to improve transfusion and clinical outcomes.
Assuntos
Plaquetas , Preservação de Sangue , DNA Mitocondrial , Selectina-P , Ativação Plaquetária , Humanos , DNA Mitocondrial/sangue , DNA Mitocondrial/metabolismo , Preservação de Sangue/métodos , Plaquetas/metabolismo , Selectina-P/metabolismo , Selectina-P/sangue , Masculino , Feminino , Contagem de Plaquetas , AdultoRESUMO
Although serum from patients with Parkinson's disease contains elevated levels of numerous pro-inflammatory cytokines including IL-6, TNF, IL-1ß, and IFNγ, whether inflammation contributes to or is a consequence of neuronal loss remains unknown1. Mutations in parkin, an E3 ubiquitin ligase, and PINK1, a ubiquitin kinase, cause early onset Parkinson's disease2,3. Both PINK1 and parkin function within the same biochemical pathway and remove damaged mitochondria from cells in culture and in animal models via mitophagy, a selective form of autophagy4. The in vivo role of mitophagy, however, is unclear, partly because mice that lack either PINK1 or parkin have no substantial Parkinson's-disease-relevant phenotypes5-7. Mitochondrial stress can lead to the release of damage-associated molecular patterns (DAMPs) that can activate innate immunity8-12, suggesting that mitophagy may mitigate inflammation. Here we report a strong inflammatory phenotype in both Prkn-/- and Pink1-/- mice following exhaustive exercise and in Prkn-/-;mutator mice, which accumulate mutations in mitochondrial DNA (mtDNA)13,14. Inflammation resulting from either exhaustive exercise or mtDNA mutation is completely rescued by concurrent loss of STING, a central regulator of the type I interferon response to cytosolic DNA15,16. The loss of dopaminergic neurons from the substantia nigra pars compacta and the motor defect observed in aged Prkn-/-;mutator mice are also rescued by loss of STING, suggesting that inflammation facilitates this phenotype. Humans with mono- and biallelic PRKN mutations also display elevated cytokines. These results support a role for PINK1- and parkin-mediated mitophagy in restraining innate immunity.
Assuntos
Imunidade Inata , Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Mitofagia , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Alarminas/metabolismo , Animais , DNA Mitocondrial/sangue , DNA Mitocondrial/genética , Humanos , Inflamação/genética , Inflamação/prevenção & controle , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/metabolismo , Condicionamento Físico Animal , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Receptor de Interferon alfa e beta/antagonistas & inibidores , Receptor de Interferon alfa e beta/imunologia , Estresse Fisiológico , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
INTRODUCTION: Epidural analgesia has been associated with intrapartum maternal fever development. Epidural-related maternal fever (ERMF) is believed to be based on a non-infectious inflammatory reaction. Circulating cell-free mitochondrial deoxyribonucleic acid (mtDNA) is one of the possible triggers of sterile inflammatory processes; however, a connection has not been investigated so far. Therefore, this study aimed to investigate cell-free mtDNA alterations in women in labour with ERMF in comparison with non-febrile women. MATERIAL AND METHODS: A total of 60 women in labour were assessed for maternal temperature every 4 h and blood samples were obtained at the beginning and after delivery. Depending on the analgesia and the development of fever (axillary temperature ≥ 37.5 °C), the women were allocated either to the group of no epidural analgesia (n = 17), to epidural analgesia no fever (n = 34) or to ERMF (n = 9). Circulating cell-free mtDNA was analysed in the maternal plasma for the primary outcome whereas secondary outcomes include the evaluation of inflammatory cytokine release, as well as placental inflammatory signs. RESULTS: Of the women with epidural analgesia, 20% (n = 9) developed ERMF and demonstrated a decrease of circulating mtDNA levels during labour (p = 0.04), but a trend towards higher free nuclear DNA. Furthermore, women with maternal pyrexia showed a 1.5 fold increased level of Interleukin-6 during labour. A correlation was found between premature rupture of membranes and ERMF. CONCLUSIONS: The pilot trial revealed an evident obstetric anaesthesia phenomenon of maternal fever due to epidural analgesia in 20% of women in labour, demonstrating counterregulated free mtDNA and nDNA. Further work is urgently required to understand the connections between the ERMF occurrence and circulating cell-free mtDNA as a potential source of sterile inflammation. TRIAL REGISTRATION: NCT0405223 on clinicaltrials.gov (registered on 25/07/2019).
Assuntos
Analgesia Epidural , DNA Mitocondrial , Febre , Humanos , Feminino , DNA Mitocondrial/sangue , Projetos Piloto , Gravidez , Adulto , Febre/sangue , Analgesia Obstétrica , Trabalho de Parto/sangue , Ácidos Nucleicos Livres/sangueRESUMO
Exposure to ionizing radiation can result in the development of a number of diseases, including cancer, cataracts and neurodegenerative pathologies. Certain occupational groups are exposed to both natural and artificial sources of radiation as a consequence of their professional activities. The development of non-invasive biomarkers to assess the risk of exposure to ionizing radiation for these groups is of great importance. In this context, our objective was to identify epigenetic and molecular biomarkers that could be used to monitor exposure to ionizing radiation. The impact of X-ray exposure on the miRNAs profile and the level of cf mtDNA were evaluated using the RT-PCR method. The levels of pro-inflammatory cytokines in their blood were quantified using the ELISA method. A significant decrease in miR-19a-3p, miR-125b-5p and significant increase in miR-29a-3p was observed in the blood plasma of individuals exposed to X-ray. High levels of pro-inflammatory cytokines and cf mtDNA were also detected. In silico identification of potential targets of these miRNAs was conducted using MIENTURNET. VDAC1 and ALOX5 were identified as possible targets. Our study identified promising biomarkers such as miRNAs and cf mtDNA that showed a dose-dependent effect of X-ray exposure.
Assuntos
Biomarcadores , DNA Mitocondrial , Epigênese Genética , MicroRNAs , Humanos , MicroRNAs/sangue , MicroRNAs/genética , DNA Mitocondrial/sangue , DNA Mitocondrial/genética , Epigênese Genética/efeitos da radiação , Biomarcadores/sangue , Raios X/efeitos adversos , Masculino , Exposição Ocupacional/efeitos adversos , Adulto , Pessoa de Meia-Idade , Citocinas/sangue , Citocinas/genética , FemininoRESUMO
Circulating cell-free DNA (ccfDNA) of mitochondrial origin (ccf-mtDNA) consists of a minor fraction of total ccfDNA in blood or in other biological fluids. Aberrant levels of ccf-mtDNA have been observed in many pathologies. Here, we introduce a simple and effective standardized Taqman probe-based dual-qPCR assay for the simultaneous detection and relative quantification of nuclear and mitochondrial fragments of ccfDNA. Three pathologies of major burden, one malignancy (Breast Cancer, BrCa), one inflammatory (Osteoarthritis, OA) and one metabolic (Type 2 Diabetes, T2D), were studied. Higher levels of ccf-mtDNA were detected both in BrCa and T2D in relation to health, but not in OA. In BrCa, hormonal receptor status was associated with ccf-mtDNA levels. Machine learning analysis of ccf-mtDNA datasets was used to build biosignatures of clinical relevance. (A) a three-feature biosignature discriminating between health and BrCa (AUC: 0.887) and a five-feature biosignature for predicting the overall survival of BrCa patients (Concordance Index: 0.756). (B) a five-feature biosignature stratifying among T2D, prediabetes and health (AUC: 0.772); a five-feature biosignature discriminating between T2D and health (AUC: 0.797); and a four-feature biosignature identifying prediabetes from health (AUC: 0.795). (C) a biosignature including total plasma ccfDNA with very high performance in discriminating OA from health (AUC: 0.934). Aberrant ccf-mtDNA levels could have diagnostic/prognostic potential in BrCa and Diabetes, while the developed multiparameter biosignatures can add value to their clinical management.
Assuntos
Neoplasias da Mama , Ácidos Nucleicos Livres , DNA Mitocondrial , Diabetes Mellitus Tipo 2 , Humanos , Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , DNA Mitocondrial/genética , Feminino , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Pessoa de Meia-Idade , Masculino , Idoso , Aprendizado de MáquinaRESUMO
Background/aim: Breast cancer is the most prevalent cancer in women, emphasizing need for noninvasive blood biomarkers to aid in treatment selection. Previous studies have demonstrated elevated levels of plasma circulating cell-free DNA (ccfDNA) in breast cancer patients. Both ccfDNA and mitochondrial DNA (mtDNA) are fragments released into the bloodstream. In this study, we investigated effectiveness of ccfDNA and mtDNA as indicators of treatment response and explored their potential as monitoring biomarkers. Additionally, we compared these markers with circulating tumor cell (CTC) data and assessed their relationship with epithelial-mesenchymal transition (EMT). Materials and methods: Thirty-six female breast cancer patients and 21 healthy females were included in the study. Quantitative polymerase chain reaction (qPCR) was performed on plasma samples to measure levels of ND1, ND4, ALU115, ALU247, and GAPDH, and DNA integrity was determined by calculating ratios of ALU247/ALU115 and ND4/ND1. Results: After treatment, patients had a significant decrease in ccfDNA levels and a significant increase in mtDNA copy number (mtDNAcn). However, there was no significant change in ccfDNA and mtDNA integrity. When comparing all groups, patients exhibited higher levels of ALU115 and ALU247 compared to controls. Moreover, patients demonstrated significantly lower ccfDNA integrity than controls. Conclusion: This study represents the first comprehensive investigation of plasma ccfDNA levels, mtDNAcn, and integrities collectively. Furthermore, it is the first study to explore the relationship between these markers and CTCs, cancer stem cell markers, treatment response, and metastatic status. Our findings suggest that plasma ccfDNA and mtDNA may serve as potential biomarkers for assessing chemotherapy response and can be employed alone or in combination with other biomarkers to monitor treatment efficacy in breast cancer patients.
Assuntos
Biomarcadores Tumorais , Neoplasias da Mama , Ácidos Nucleicos Livres , DNA Mitocondrial , Transição Epitelial-Mesenquimal , Células Neoplásicas Circulantes , Humanos , Feminino , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , DNA Mitocondrial/sangue , Células Neoplásicas Circulantes/patologia , Pessoa de Meia-Idade , Ácidos Nucleicos Livres/sangue , Biomarcadores Tumorais/sangue , Adulto , Terapia Neoadjuvante , IdosoRESUMO
The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation fueled by damage associated molecular patterns (DAMPs). We show that elevated cell-free DNA (cfDNA) in patients with SCD is not just a prognostic biomarker, it also contributes to the pathological inflammation. Within the elevated cfDNA, patients with SCD had a significantly higher ratio of cell-free mitochondrial DNA (cf-mtDNA)/cell-free nuclear DNA compared with healthy controls. Additionally, mitochondrial DNA in patient samples showed significantly disproportionately increased hypomethylation compared with healthy controls, and it was increased further in crises compared with steady-state. Using flow cytometry, structured illumination microscopy, and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria and, thus, are likely to be the source of the elevated cf-mtDNA in patients with SCD. Patient plasma containing high levels of cf-mtDNA triggered the formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1, implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic DAMP, highlighting an underappreciated role for mitochondria in sickle pathology. These trials were registered at www.clinicaltrials.gov as #NCT00081523, #NCT03049475, and #NCT00047996.
Assuntos
Anemia Falciforme/sangue , Ácidos Nucleicos Livres/sangue , Metilação de DNA , DNA Mitocondrial/sangue , Adulto , Idoso , Biomarcadores/sangue , Armadilhas Extracelulares/metabolismo , Feminino , Humanos , Inflamação/sangue , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Nucleotidiltransferases/metabolismo , Transdução de SinaisRESUMO
Circulating cell-free mitochondrial DNA (ccf-mtDNA) may induce systemic inflammation, a common condition in chronic kidney disease (CKD), by acting as a damage-associated molecular pattern. We hypothesized that in patients with moderate to severe CKD, aerobic exercise would reduce ccf-mtDNA levels. We performed a post hoc analysis of a multicenter randomized trial (NCT01150851) measuring plasma concentrations of ccf-mtDNA at baseline and 2 and 4 mo after aerobic exercise and caloric restriction. A total of 99 participants had baseline ccf-mtDNA, and 92 participants completed the study. The median age of the participants was 57 yr, 44% were female and 55% were male, 23% had diabetes, and 92% had hypertension. After adjusting for demographics, blood pressure, body mass index, diabetes, and estimated glomerular filtration rate, median ccf-mtDNA concentrations at baseline, 2 mo, and 4 mo were 3.62, 3.08, and 2.78 pM for the usual activity group and 2.01, 2.20, and 2.67 pM for the aerobic exercise group, respectively. A 16.1% greater increase per month in ccf-mtDNA was seen in aerobic exercise versus usual activity (P = 0.024), which was more pronounced with the combination of aerobic exercise and caloric restriction (29.5% greater increase per month). After 4 mo of intervention, ccf-mtDNA increased in the aerobic exercise group by 81.6% (95% confidence interval: 8.2-204.8, P = 0.024) compared with the usual activity group and was more marked in the aerobic exercise and caloric restriction group (181.7% increase, 95% confidence interval: 41.1-462.2, P = 0.003). There was no statistically significant correlation between markers of oxidative stress and inflammation with ccf-mtDNA. Our data indicate that aerobic exercise increased ccf-mtDNA levels in patients with moderate to severe CKD.NEW & NOTEWORTHY The effects of prolonged exercise on circulating cell-free mitochondrial DNA (ccf-mtDNA) have not been explored in patients with chronic kidney disease (CKD). We showed that 4-mo aerobic exercise is associated with an increase in plasma ccf-mtDNA levels in patients with stages 3 or 4 CKD. These changes were not associated with markers of systemic inflammation. Future studies should determine the mechanisms by which healthy lifestyle interventions influence biomarkers of inflammation and oxidative stress in patients with CKD.
Assuntos
Restrição Calórica , Ácidos Nucleicos Livres/genética , DNA Mitocondrial/genética , Exercício Físico , Estilo de Vida Saudável , Insuficiência Renal Crônica/terapia , Idoso , Biomarcadores/sangue , Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , Feminino , Humanos , Mediadores da Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Projetos Piloto , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/genética , Índice de Gravidade de Doença , Fatores de Tempo , Resultado do Tratamento , Estados Unidos , Regulação para CimaRESUMO
BACKGROUND: Mitochondrial genome copy number (MT-CN) varies among humans and across tissues and is highly heritable, but its causes and consequences are not well understood. When measured by bulk DNA sequencing in blood, MT-CN may reflect a combination of the number of mitochondria per cell and cell-type composition. Here, we studied MT-CN variation in blood-derived DNA from 19184 Finnish individuals using a combination of genome (N = 4163) and exome sequencing (N = 19034) data as well as imputed genotypes (N = 17718). RESULTS: We identified two loci significantly associated with MT-CN variation: a common variant at the MYB-HBS1L locus (P = 1.6 × 10-8), which has previously been associated with numerous hematological parameters; and a burden of rare variants in the TMBIM1 gene (P = 3.0 × 10-8), which has been reported to protect against non-alcoholic fatty liver disease. We also found that MT-CN is strongly associated with insulin levels (P = 2.0 × 10-21) and other metabolic syndrome (metS)-related traits. Using a Mendelian randomization framework, we show evidence that MT-CN measured in blood is causally related to insulin levels. We then applied an MT-CN polygenic risk score (PRS) derived from Finnish data to the UK Biobank, where the association between the PRS and metS traits was replicated. Adjusting for cell counts largely eliminated these signals, suggesting that MT-CN affects metS via cell-type composition. CONCLUSION: These results suggest that measurements of MT-CN in blood-derived DNA partially reflect differences in cell-type composition and that these differences are causally linked to insulin and related traits.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/sangue , Proteínas de Ligação ao GTP/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas c-myb/genética , Adulto , Idoso , Linhagem da Célula/genética , DNA Mitocondrial/genética , Feminino , Predisposição Genética para Doença , Genoma Mitocondrial/genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Análise da Randomização Mendeliana , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Sequenciamento do ExomaRESUMO
Among cardiovascular disease (CVD) biomarkers, the mitochondrial DNA copy number (mtDNAcn) is a promising candidate. A growing attention has been also dedicated to trimethylamine-N-oxide (TMAO), an oxidative derivative of the gut metabolite trimethylamine (TMA). With the aim to identify biomarkers predictive of CVD, we investigated TMA, TMAO, and mtDNAcn in a population of 389 coronary artery disease (CAD) patients and 151 healthy controls, in association with established risk factors for CVD (sex, age, hypertension, smoking, diabetes, glomerular filtration rate [GFR]) and troponin, an established marker of CAD. MtDNAcn was significantly lower in CAD patients; it correlates with GFR and TMA, but not with TMAO. A biomarker including mtDNAcn, sex, and hypertension (but neither TMA nor TMAO) emerged as a good predictor of CAD. Our findings support the mtDNAcn as a promising plastic biomarker, useful to monitor the exposure to risk factors and the efficacy of preventive interventions for a personalized CAD risk reduction.
Assuntos
Biomarcadores/sangue , Doenças Cardiovasculares/diagnóstico , Variações do Número de Cópias de DNA , DNA Mitocondrial/sangue , Trato Gastrointestinal/metabolismo , Metilaminas/sangue , Idoso , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/genética , Estudos de Casos e Controles , Estudos de Coortes , DNA Mitocondrial/genética , Feminino , Humanos , Masculino , Fatores de RiscoRESUMO
The measurement of the level of mitochondrial DNA (mtDNA) in the blood is a difficult problem due to high variability of mitochondrial genes, deletions in the mitochondrial genome in some pathological conditions, different sources of mtDNA into the bloodstream (mtDNA from tissues, from blood cells, etc.). We designed primers and TaqMan probes for highly conserved regions of the ND1 and ND2 genes outside the mitochondrial deletions "hot zones". For standardizing the technique, the true concentration of low-molecular-weight mtDNA was determined by real-time PCR for two targets: a fragment of the ND2 gene (122 bp) and the ND1 and ND2 genes (1198 bp). The sensitivity and specificity of the developed approach were verified on a DNA pool isolated from the blood plasma of healthy donors of various nationalities. The concentration of low-molecular-weight mtDNA in the blood plasma of two patients with COVID-19 was monitored over two weeks of inpatient treatment. A significant increase in the content of low-molecular-weight mtDNA was observed during the first 5 days after hospitalization, followed by a drop to the level of healthy donors. The developed technique makes it possible to assess the blood level of low-molecular-weight mtDNA regardless of the quality of sampling and makes it possible to standardize this biological marker in a wide range of infectious and non-infectious pathologies.
Assuntos
COVID-19/metabolismo , Ácidos Nucleicos Livres/genética , DNA Mitocondrial/genética , NADH Desidrogenase/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Adulto , Idoso , COVID-19/virologia , Estudos de Casos e Controles , Ácidos Nucleicos Livres/sangue , Primers do DNA/síntese química , DNA Mitocondrial/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/virologia , NADH Desidrogenase/sangue , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/patogenicidadeRESUMO
BACKGROUND: Combination antiretroviral therapy (cART) during pregnancy prevents vertical transmission, but many antiretrovirals cross the placenta and several can affect mitochondria. Exposure to maternal human immunodeficiency virus (HIV) and/or cART could have long-term effects on children who are HIV exposed and uninfected (CHEU). Our objective was to compare blood mitochondrial DNA (mtDNA) content in CHEU and children who are HIV unexposed and uninfected (CHUU), at birth and in early life. METHODS: Whole-blood mtDNA content at birth and in early life (age 0-3 years) was compared cross-sectionally between CHEU and CHUU. Longitudinal changes in mtDNA content among CHEU was also evaluated. RESULTS: At birth, CHEU status and younger gestational age were associated with higher mtDNA content. These remained independently associated with mtDNA content in multivariable analyses, whether considering all infants, or only those born at term. Longitudinally, CHEU mtDNA levels remained unchanged during the first 6 months of life, and gradually declined thereafter. A separate age- and sex-matched cross-sectional analysis (in 214 CHEU and 214 CHUU) illustrates that the difference in mtDNA between the groups remains detectable throughout the first 3 years of life. CONCLUSION: The persistently elevated blood mtDNA content observed among CHEU represents a long-term effect, possibly resulting from in utero stresses related to maternal HIV and/or cART. The clinical impact of altered mtDNA levels is unclear.
Assuntos
Fármacos Anti-HIV/uso terapêutico , DNA Mitocondrial/sangue , Infecções por HIV/tratamento farmacológico , Complicações Infecciosas na Gravidez/tratamento farmacológico , Efeitos Tardios da Exposição Pré-Natal , Terapia Antirretroviral de Alta Atividade , Pré-Escolar , Estudos Transversais , Feminino , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , Humanos , Lactente , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Estudos Longitudinais , Masculino , GravidezRESUMO
Obesity is associated with chronic low-grade inflammation that eventually leads to metabolic complications. Extracellular DNA (ecDNA) is a damage-associated molecular pattern. Extracellular mitochondrial DNA can activate innate immunity. We hypothesized that ecDNA, especially of mitochondrial origin, could be associated with components of the metabolic syndrome in young healthy probands. In a cross-sectional study, healthy adolescents (n = 1,249) provided blood samples. Anthropometric data, blood pressure, and blood counts were assessed. In addition, biochemical analysis of sera or plasma was conducted, including the quantification of advanced oxidation protein products (AOPPs) as a marker of oxidative stress induced by neutrophil or monocyte activation. Plasma ecDNA was isolated and measured by fluorometry. Nuclear and mitochondrial DNA were quantified by real-time PCR. Males had higher total plasma ecDNA [15 (11-21) vs. 11 (8-17) ng/mL; median (interquartile range)], nuclear [1,760 (956-3,273) vs. 1,153 (600-2,292) genome equivalents (GE)/mL], and mitochondrial [37,181 (14,836-90,896) vs. 30,089 (12,587-72,286) GE/mL] DNA. ecDNA correlated positively with the continuous metabolic syndrome score (r = 0.158 for males and r = 0.134 for females). Stronger correlations were found between ecDNA of mitochondrial origin and AOPP (r = 0.202 and 0.186 for males and females, respectively). Multivariate regression analysis revealed associations of nuclear DNA with leukocyte and erythrocyte counts. The results of this study of healthy adolescents show that circulating ecDNA is associated with the risk of metabolic syndrome, not with obesity per se. The association between mitochondrial ecDNA and AOPP requires further attention as it supports a potential role of mitochondria-induced sterile inflammation in the pathogenesis of the metabolic syndrome.
Assuntos
Ácidos Nucleicos Livres/sangue , Síndrome Metabólica/sangue , Síndrome Metabólica/genética , Adolescente , Produtos da Oxidação Avançada de Proteínas/sangue , Biomarcadores/sangue , Pressão Sanguínea , Criança , Estudos Transversais , DNA Mitocondrial/sangue , Feminino , Humanos , Masculino , Síndrome Metabólica/epidemiologia , Prevalência , Análise de Regressão , Fatores de Risco , Eslováquia/epidemiologia , Adulto JovemRESUMO
Progress in the study of circulating, cell-free nuclear DNA (ccf-nDNA) in cancer detection has led to the development of noninvasive clinical diagnostic tests and has accelerated the evaluation of ccf-nDNA abundance as a disease biomarker. Likewise, circulating, cell-free mitochondrial DNA (ccf-mtDNA) is under similar investigation. However, optimal ccf-mtDNA isolation parameters have not been established, and inconsistent protocols for ccf-nDNA collection, storage, and analysis have hindered its clinical utility. Until now, no studies have established a method for high-throughput isolation that considers both ccf-nDNA and ccf-mtDNA. We initially optimized human plasma digestion and extraction conditions for maximal recovery of these DNAs using a magnetic bead-based isolation method. However, when we incorporated this method onto a high-throughput platform, initial experiments found that DNA isolated from identical human plasma samples displayed plate edge effects resulting in low ccf-mtDNA reproducibility, whereas ccf-nDNA was less affected. Therefore, we developed a detailed protocol optimized for both ccf-mtDNA and ccf-nDNA recovery that uses a magnetic bead-based isolation process on an automated 96-well platform. Overall, we calculate an improved efficiency of recovery of â¼95-fold for ccf-mtDNA and 20-fold for ccf-nDNA when compared with the initial procedure. Digestion conditions, liquid-handling characteristics, and magnetic particle processor programming all contributed to increased recovery without detectable positional effects. To our knowledge, this is the first high-throughput approach optimized for ccf-mtDNA and ccf-nDNA recovery and serves as an important starting point for clinical studies.
Assuntos
Núcleo Celular/genética , Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , Ensaios de Triagem em Larga Escala/métodos , Mitocôndrias/genética , Automação , Ácidos Nucleicos Livres/isolamento & purificação , Ácidos Nucleicos Livres/metabolismo , DNA Mitocondrial/isolamento & purificação , DNA Mitocondrial/metabolismo , Endopeptidase K/metabolismo , Humanos , Magnetismo , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real , TemperaturaRESUMO
The effect of mitochondrial DNA (mtDNA) variation on peripheral blood transcriptomics in health and disease is not fully known. Sex-specific mitochondrially controlled gene expression patterns have been shown in Drosophila melanogaster but in humans, evidence is lacking. Functional variation in mtDNA may also have a role in the development of type 2 diabetes and its precursor state, i.e. prediabetes. We examined the associations between mitochondrial single-nucleotide polymorphisms (mtSNPs) and peripheral blood transcriptomics with a focus on sex- and prediabetes-specific effects. The genome-wide blood cell expression data of 19 637 probes, 199 deep-sequenced mtSNPs and nine haplogroups of 955 individuals from a population-based Young Finns Study cohort were used. Significant associations were identified with linear regression and analysis of covariance. The effects of sex and prediabetes on the associations between gene expression and mtSNPs were studied using random-effect meta-analysis. Our analysis identified 53 significant expression probe-mtSNP associations after Bonferroni correction, involving 7 genes and 31 mtSNPs. Eight probe-mtSNP signals remained independent after conditional analysis. In addition, five genes showed differential expression between haplogroups. The meta-analysis did not show any significant differences in linear model effect sizes between males and females but identified the association between the OASL gene and mtSNP C16294T to show prediabetes-specific effects. This study pinpoints new independent mtSNPs associated with peripheral blood transcriptomics and replicates six previously reported associations, providing further evidence of the mitochondrial genetic control of blood cell gene expression. In addition, we present evidence that prediabetes might lead to perturbations in mitochondrial control.
Assuntos
DNA Mitocondrial/genética , Regulação da Expressão Gênica/genética , Adulto , Sequência de Bases , Células Sanguíneas/metabolismo , Células Sanguíneas/fisiologia , DNA Mitocondrial/sangue , Diabetes Mellitus Tipo 2/genética , Feminino , Expressão Gênica , Estudos de Associação Genética/métodos , Variação Genética/genética , Genética Populacional/métodos , Estudo de Associação Genômica Ampla/métodos , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Transcriptoma/genéticaRESUMO
Sepsis remains a leading cause of mortality in critically ill patients and is characterized by multi-organ dysfunction. Mitochondrial damage has been proposed to be involved in the pathophysiology of sepsis. In addition to metabolic impairments resulting from mitochondrial dysfunction, mitochondrial DNA (mtDNA) causes systemic inflammation as a damage-associated molecular pattern when it is released to the circulation. Metabolic derangements in skeletal muscle are a major complication of sepsis and negatively affects clinical outcomes of septic patients. However, limited knowledge is available about sepsis-induced mitochondrial damage in skeletal muscle. Here, we show that sepsis induced profound abnormalities in cristae structure, rupture of the inner and outer membranes and enlargement of the mitochondria in mouse skeletal muscle in a time-dependent manner, which was associated with increased plasma mtDNA levels. Farnesyltransferase inhibitor, FTI-277, prevented sepsis-induced morphological aberrations of the mitochondria, and blocked the increased plasma mtDNA levels along with improved survival. These results indicate that protein farnesylation plays a role in sepsis-induced damage of the mitochondria in mouse skeletal muscle. Our findings suggest that mitochondrial disintegrity in skeletal muscle may contribute to elevated circulating mtDNA levels in sepsis.
Assuntos
DNA Mitocondrial/sangue , Farnesiltranstransferase/antagonistas & inibidores , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Sepse/tratamento farmacológico , Animais , Masculino , Metionina/análogos & derivados , Metionina/farmacologia , Camundongos , Mitocôndrias/patologia , Músculo Esquelético/patologia , Sepse/sangue , Sepse/patologia , Fatores de TempoRESUMO
Hepatic injury is common in patients who suffer from severe burns plus delayed resuscitation (B + DR). Stimulator of interferon genes (STING) is primarily expressed in Kupffer cells (KCs). We demonstrated that B + DR caused hepatic injury and oxidative stress. Reactive oxygen species (ROS) damage mitochondrial membranes in hepatocytes, leading to the release of mitochondrial DNA (mtDNA) into the hepatocyte cytosol and the circulation. The damaged hepatocytes then activate the mtDNA/STING pathway in KCs and trigger KCs polarization towards pro-inflammatory phenotype. SS-31 is a strong antioxidant that specifically concentrates in the inner mitochondrial membrane. SS-31 prevented hepatic injury by neutralizing ROS, inhibiting the release of mtDNA, protecting hepatocyte mitochondria, suppressing the activation of the mtDNA/STING pathway and inhibiting KCs polarization into pro-inflammatory phenotype.