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The risk of early recurrent events after stroke remains high despite currently established secondary prevention strategies1. Risk is particularly high in patients with atherosclerosis, with more than 10% of patients experiencing early recurrent events1,2. However, despite the enormous medical burden of this clinical phenomenon, the underlying mechanisms leading to increased vascular risk and recurrent stroke are largely unknown. Here, using a novel mouse model of stroke-induced recurrent ischaemia, we show that stroke leads to activation of the AIM2 inflammasome in vulnerable atherosclerotic plaques via an increase of circulating cell-free DNA. Enhanced plaque inflammation post-stroke results in plaque destabilization and atherothrombosis, finally leading to arterioarterial embolism and recurrent stroke within days after the index stroke. We confirm key steps of plaque destabilization also after experimental myocardial infarction and in carotid artery plaque samples from patients with acute stroke. Rapid neutrophil NETosis was identified as the main source of cell-free DNA after stroke and NET-DNA as the causative agent leading to AIM2 inflammasome activation. Neutralization of cell-free DNA by DNase treatment or inhibition of inflammasome activation reduced the rate of stroke recurrence after experimental stroke. Our findings present an explanation for the high recurrence rate after incident ischaemic events in patients with atherosclerosis. The detailed mechanisms uncovered here provide clinically uncharted therapeutic targets for which we show high efficacy to prevent recurrent events. Targeting DNA-mediated inflammasome activation after remote tissue injury represents a promising avenue for further clinical development in the prevention of early recurrent events.
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OBJECTIVE: To present a novel method that uses an epigenetic fingerprint to measure changes in plasma concentrations of cardiac-specific cell-free DNA (CS-cfDNA) as a marker of myocardial cell death. METHODS: This prospective, analytic, observational comparative study included patients with heart disease or multiple risk factors for heart disease undergoing major noncardiac, mostly vascular surgery, requiring an arterial-line, and at least 24 h hospitalization in the post anaesthesia care unit or critical care unit after surgery. Blood samples were collected at least four times per patient to measure troponin-T (via high-sensitivity troponin-T test) and CS-cfDNA pre- and postoperatively. RESULTS: A total of 117 patients were included (group 1, 77 patients [66%] with low preoperative and postoperative troponin-T; group 2, 18 patients [15%] with low preoperative but increased postoperative troponin-T; group 3, 16 patients [14%] with high troponin-T both preoperatively and postoperatively; and group 4, six patients [5%] with elevated preoperative troponin-T that decreased postoperatively). The increase in CS-cfDNA after surgery was statistically significant only in group 2, which correlated with an increase in troponin-T in the same group. CONCLUSIONS: CS-cfDNA increased early postoperatively, particularly in patients with silent postoperative troponin elevation, and was correlated with an increase in troponin-T. These results may suggest that, in the subgroup of patients with postoperative elevated troponin, cardiomyocyte death indeed occurred.
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Ácidos Nucleicos Livres , Troponina T , Humanos , Biomarcadores , DNA , Estudos Prospectivos , Procedimentos Cirúrgicos Vasculares/efeitos adversos , Infarto do Miocárdio , Complicações Pós-OperatóriasRESUMO
Assessment of pancreas cell type composition is crucial to the understanding of the genesis of diabetes. Current approaches use immunodetection of protein markers, for example, insulin as a marker of ß-cells. A major limitation of these methods is that protein content varies in physiological and pathological conditions, complicating the extrapolation to actual cell number. Here, we demonstrate the use of cell type-specific DNA methylation markers for determining the fraction of specific cell types in human islet and pancreas specimens. We identified genomic loci that are uniquely demethylated in specific pancreatic cell types and applied targeted PCR to assess the methylation status of these loci in tissue samples, enabling inference of cell type composition. In islet preparations, normalization of insulin secretion to ß-cell DNA revealed similar ß-cell function in pre-type 1 diabetes (T1D), T1D, and type 2 diabetes (T2D), which was significantly lower than in donors without diabetes. In histological pancreas specimens from recent-onset T1D, this assay showed ß-cell fraction within the normal range, suggesting a significant contribution of ß-cell dysfunction. In T2D pancreata, we observed increased α-cell fraction and normal ß-cell fraction. Methylation-based analysis provides an accurate molecular alternative to immune detection of cell types in the human pancreas, with utility in the interpretation of insulin secretion assays and the assessment of pancreas cell composition in health and disease.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Células Secretoras de Glucagon , Células Secretoras de Insulina , Ilhotas Pancreáticas , Humanos , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ilhotas Pancreáticas/metabolismo , Metilação de DNA , Pâncreas/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Glucagon/metabolismoRESUMO
BACKGROUND: Accurate detection of graft-versus-host disease (GVHD) is a major challenge in the management of patients undergoing hematopoietic stem cell transplantation (HCT). Here, we demonstrated the use of circulating cell-free DNA (cfDNA) for detection of tissue turnover and chronic GVHD (cGVHD) in specific organs. METHODS: We established a cocktail of tissue-specific DNA methylation markers and used it to determine the concentration of cfDNA molecules derived from the liver, skin, lungs, colon, and specific immune cells in 101 patients undergoing HCT. RESULTS: Patients with active cGVHD showed elevated concentrations of cfDNA, as well as tissue-specific methylation markers that agreed with clinical scores. Strikingly, transplanted patients with no clinical symptoms had abnormally high levels of tissue-specific markers, suggesting hidden tissue turnover even in the absence of evident clinical pathology. An integrative model taking into account total cfDNA concentration, monocyte/macrophage cfDNA levels and alanine transaminase was able to correctly identify GVHD with a specificity of 86% and precision of 89% (AUC of 0.8). CONCLUSION: cfDNA markers can be used for the detection of cGVHD, opening a window into underlying tissue dynamics in patients that receive allogeneic stem cell transplants. FUNDING: This work was supported by grants from the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation and the Helmsley Charitable Trust (to YD).
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Síndrome de Bronquiolite Obliterante , Ácidos Nucleicos Livres , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Metilação de DNA , Ácidos Nucleicos Livres/genética , Doença Enxerto-Hospedeiro/diagnóstico , Biomarcadores , Marcadores Genéticos , Doença CrônicaRESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed at an advanced stage. Liquid biopsy approaches may facilitate detection of early stage PDAC when curative treatments can be employed. DESIGN: To assess circulating marker discrimination in training, testing and validation patient cohorts (total n=426 patients), plasma markers were measured among PDAC cases and patients with chronic pancreatitis, colorectal cancer (CRC), and healthy controls. Using CA19-9 as an anchor marker, measurements were made of two protein markers (TIMP1, LRG1) and cell-free DNA (cfDNA) pancreas-specific methylation at 9 loci encompassing 61 CpG sites. RESULTS: Comparative methylome analysis identified nine loci that were differentially methylated in exocrine pancreas DNA. In the training set (n=124 patients), cfDNA methylation markers distinguished PDAC from healthy and CRC controls. In the testing set of 86 early stage PDAC and 86 matched healthy controls, CA19-9 had an area under the receiver operating characteristic curve (AUC) of 0.88 (95% CI 0.83 to 0.94), which was increased by adding TIMP1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.06), LRG1 (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02) or exocrine pancreas-specific cfDNA methylation markers at nine loci (AUC 0.92; 95% CI 0.88 to 0.96; p=0.02). In the validation set of 40 early stage PDAC and 40 matched healthy controls, a combined panel including CA19-9, TIMP1 and a 9-loci cfDNA methylation panel had greater discrimination (AUC 0.86, 95% CI 0.77 to 0.95) than CA19-9 alone (AUC 0.82; 95% CI 0.72 to 0.92). CONCLUSION: A combined panel of circulating markers including proteins and methylated cfDNA increased discrimination compared with CA19-9 alone for early stage PDAC.
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Adenocarcinoma , Carcinoma Ductal Pancreático , Ácidos Nucleicos Livres , Neoplasias Pancreáticas , Humanos , Antígeno CA-19-9 , Biomarcadores Tumorais , Ácidos Nucleicos Livres/metabolismo , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Pâncreas/patologia , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Metilação de DNARESUMO
BACKGROUND: Radiotherapy has an important role in the treatment of brain metastases but carries risk of short and/or long-term toxicity, termed radiation-induced brain injury (RBI). As the diagnosis of RBI is crucial for correct patient management, there is an unmet need for reliable biomarkers for RBI. The aim of this proof-of concept study is to determine the utility of brain-derived circulating free DNA (BncfDNA), identified by specific methylation patterns for neurons, astrocytes, and oligodendrocytes, as biomarkers brain injury induced by radiotherapy. METHODS: Twenty-four patients with brain metastases were monitored clinically and radiologically before, during and after brain radiotherapy, and blood for BncfDNA analysis (98 samples) was concurrently collected. Sixteen patients were treated with whole brain radiotherapy and eight patients with stereotactic radiosurgery. RESULTS: During follow-up nine RBI events were detected, and all correlated with significant increase in BncfDNA levels compared to baseline. Additionally, resolution of RBI correlated with a decrease in BncfDNA. Changes in BncfDNA were independent of tumor response. CONCLUSIONS: Elevated BncfDNA levels reflects brain cell injury incurred by radiotherapy. further research is needed to establish BncfDNA as a novel plasma-based biomarker for brain injury induced by radiotherapy.
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Lesões Encefálicas , Neoplasias Encefálicas , Lesões por Radiação , Radiocirurgia , Humanos , Projetos Piloto , Encéfalo , Neoplasias Encefálicas/secundário , Lesões Encefálicas/etiologia , Lesões Encefálicas/cirurgia , Lesões por Radiação/etiologiaRESUMO
Circulating cell-free DNA (cfDNA) fragments are a biological analyte with extensive utility in diagnostic medicine. Understanding the source of cfDNA and mechanisms of release is crucial for designing and interpreting cfDNA-based liquid biopsy assays. Using cell type-specific methylation markers as well as genome-wide methylation analysis, we determine that megakaryocytes, the precursors of anuclear platelets, are major contributors to cfDNA (~26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Surprisingly, we discover that platelets contain genomic DNA fragments originating in megakaryocytes, contrary to the general understanding that platelets lack genomic DNA. Megakaryocyte-derived cfDNA is increased in pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased upon reduced platelet production due to chemotherapy-induced bone marrow suppression. Similarly, erythroblast cfDNA is reflective of erythrocyte production and is elevated in patients with thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns can thus serve as biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis, which can aid in determining the etiology of aberrant levels of erythrocytes and platelets.
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Ácidos Nucleicos Livres , Megacariócitos , Humanos , Trombopoese , Eritropoese/genética , Ácidos Nucleicos Livres/genética , Plaquetas , Eritroblastos , DNARESUMO
Chronological age prediction from DNA methylation sheds light on human aging, health, and lifespan. Current clocks are mostly based on linear models and rely upon hundreds of sites across the genome. Here, we present GP-age, an epigenetic non-linear cohort-based clock for blood, based upon 11,910 methylomes. Using 30 CpG sites alone, GP-age outperforms state-of-the-art models, with a median accuracy of â¼2 years on held-out blood samples, for both array and sequencing-based data. We show that aging-related changes occur at multiple neighboring CpGs, with implications for using fragment-level analysis of sequencing data in aging research. By training three independent clocks, we show enrichment of donors with consistent deviation between predicted and actual age, suggesting individual rates of biological aging. Overall, we provide a compact yet accurate alternative to array-based clocks for blood, with applications in longitudinal aging research, forensic profiling, and monitoring epigenetic processes in transplantation medicine and cancer.
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Envelhecimento , Metilação de DNA , Humanos , Pré-Escolar , Metilação de DNA/genética , Envelhecimento/genética , Algoritmos , Sequência de Bases , Epigênese GenéticaRESUMO
BACKGROUND: Donor hyperglycaemia following brain death has been attributed to reversible insulin resistance. However, our islet and pancreas transplant data suggest that other mechanisms may be predominant. We aimed to determine the relationships between donor insulin use and markers of beta-cell death and beta-cell function in pancreas donors after brain death. METHODS: In pancreas donors after brain death, we compared clinical and biochemical data in 'insulin-treated' and 'not insulin-treated donors' (IT vs. not-IT). We measured plasma glucose, C-peptide and levels of circulating unmethylated insulin gene promoter cell-free DNA (INS-cfDNA) and microRNA-375 (miR-375), as measures of beta-cell death. Relationships between markers of beta-cell death and islet isolation outcomes and post-transplant function were also evaluated. RESULTS: Of 92 pancreas donors, 40 (43%) required insulin. Glycaemic control and beta-cell function were significantly poorer in IT donors versus not-IT donors [median (IQR) peak glucose: 8 (7-11) vs. 6 (6-8) mmol/L, p = .016; C-peptide: 3280 (3159-3386) vs. 3195 (2868-3386) pmol/L, p = .046]. IT donors had significantly higher levels of INS-cfDNA [35 (18-52) vs. 30 (8-51) copies/ml, p = .035] and miR-375 [1.050 (0.19-1.95) vs. 0.73 (0.32-1.10) copies/nl, p = .05]. Circulating donor miR-375 was highly predictive of recipient islet graft failure at 3 months [adjusted receiver operator curve (SE) = 0.813 (0.149)]. CONCLUSIONS: In pancreas donors, hyperglycaemia requiring IT is strongly associated with beta-cell death. This provides an explanation for the relationship of donor IT with post-transplant beta-cell dysfunction in transplant recipients.
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Ácidos Nucleicos Livres , Hiperglicemia , Transplante das Ilhotas Pancreáticas , MicroRNAs , Humanos , Peptídeo C , Morte Encefálica , Insulina/genética , Doadores de Tecidos , Morte CelularRESUMO
Strenuous physical exercise causes a massive elevation in the concentration of circulating cell-free DNA (cfDNA), which correlates with effort intensity and duration. The cellular sources and physiological drivers of this phenomenon are unknown. Using methylation patterns of cfDNA and associated histones, we show that cfDNA in exercise originates mostly in extramedullary polymorphonuclear neutrophils. Strikingly, cardiomyocyte cfDNA concentration increases after a marathon, consistent with elevated troponin levels and indicating low-level, delayed cardiac cell death. Physical impact, low oxygen levels, and elevated core body temperature contribute to neutrophil cfDNA release, while muscle contraction, increased heart rate, ß-adrenergic signaling, or steroid treatment fail to cause elevation of cfDNA. Physical training reduces neutrophil cfDNA release after a standard exercise, revealing an inverse relationship between exercise-induced cfDNA release and training level. We speculate that the release of cfDNA from neutrophils in exercise relates to the activation of neutrophils in the context of exercise-induced muscle damage.
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Ácidos Nucleicos Livres , Neutrófilos , Miócitos Cardíacos , Exercício Físico/fisiologia , HistonasRESUMO
BACKGROUND: Vascular endothelial cells (VECs) are an essential component of each tissue, contribute to multiple pathologies, and are targeted by important drugs. Yet, there is a shortage of biomarkers to assess VEC turnover. METHODS: To develop DNA methylation-based liquid biopsies for VECs, we determined the methylome of VECs isolated from freshly dissociated human tissues. FINDINGS: A comparison with a human cell-type methylome atlas yielded thousands of loci that are uniquely unmethylated in VECs. These sites are typically gene enhancers, often residing adjacent to VEC-specific genes. We also identified hundreds of genomic loci that are differentially methylated in organotypic VECs, indicating that VECs feeding specific organs are distinct cell types with a stable epigenetic identity. We established universal and lung-specific VEC markers and evaluated their presence in circulating cell-free DNA (cfDNA). Nearly 2.5% of cfDNA in the plasma of healthy individuals originates from VECs. Sepsis, graft versus host disease, and cardiac catheterization are associated with elevated levels of VEC-derived cfDNA, indicative of vascular damage. Lung-specific VEC cfDNA is selectively elevated in patients with chronic obstructive pulmonary disease (COPD) or lung cancer, revealing tissue-specific vascular turnover. CONCLUSIONS: VEC cfDNA biomarkers inform vascular dynamics in health and disease, potentially contributing to early diagnosis and monitoring of pathologies, and assessment of drug activity. FUNDING: This work was supported by the Beutler Research Program, Helmsley Charitable Trust, JDRF, Grail and the DON Foundation (to Y.D.). Y.D holds the Walter & Greta Stiel Chair in heart studies. B.G., R.S., J.M., D.N., T.K., and Y.D. filed patents on cfDNA analysis.
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Ácidos Nucleicos Livres , Epigenoma , Humanos , Endotélio Vascular , Células Endoteliais/metabolismo , Biomarcadores/metabolismo , Biópsia LíquidaRESUMO
Immune and inflammatory processes occurring within tissues are often undetectable by blood cell counts, standard circulating biomarkers, or imaging, representing an unmet biomedical need. Here, we outline recent advances indicating that liquid biopsies can broadly inform human immune system dynamics. Nucleosome-size fragments of cell-free DNA (cfDNA) released from dying cells into blood contain rich epigenetic information such as methylation, fragmentation, and histone mark patterns. This information allows to infer the cfDNA cell of origin, as well as pre-cell death gene expression patterns. We propose that the analysis of epigenetic features of immune cell-derived cfDNA can shed light on immune cell turnover dynamics in healthy people, and inform the study and diagnosis of cancer, local inflammation, infectious or autoimmune diseases, as well as responses to vaccination.
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Ácidos Nucleicos Livres , Metilação de DNA , Humanos , Biópsia Líquida/métodos , Biomarcadores , Ácidos Nucleicos Livres/genética , Inflamação/genética , Epigênese GenéticaRESUMO
DNA methylation is a fundamental epigenetic mark that governs gene expression and chromatin organization, thus providing a window into cellular identity and developmental processes1. Current datasets typically include only a fraction of methylation sites and are often based either on cell lines that underwent massive changes in culture or on tissues containing unspecified mixtures of cells2-5. Here we describe a human methylome atlas, based on deep whole-genome bisulfite sequencing, allowing fragment-level analysis across thousands of unique markers for 39 cell types sorted from 205 healthy tissue samples. Replicates of the same cell type are more than 99.5% identical, demonstrating the robustness of cell identity programmes to environmental perturbation. Unsupervised clustering of the atlas recapitulates key elements of tissue ontogeny and identifies methylation patterns retained since embryonic development. Loci uniquely unmethylated in an individual cell type often reside in transcriptional enhancers and contain DNA binding sites for tissue-specific transcriptional regulators. Uniquely hypermethylated loci are rare and are enriched for CpG islands, Polycomb targets and CTCF binding sites, suggesting a new role in shaping cell-type-specific chromatin looping. The atlas provides an essential resource for study of gene regulation and disease-associated genetic variants, and a wealth of potential tissue-specific biomarkers for use in liquid biopsies.
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Células , Metilação de DNA , Epigênese Genética , Epigenoma , Humanos , Linhagem Celular , Células/classificação , Células/metabolismo , Cromatina/genética , Cromatina/metabolismo , Ilhas de CpG/genética , DNA/genética , DNA/metabolismo , Desenvolvimento Embrionário , Elementos Facilitadores Genéticos , Especificidade de Órgãos , Proteínas do Grupo Polycomb/metabolismo , Sequenciamento Completo do GenomaRESUMO
The standard treatment approach for stage II/III rectal cancer is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. In recent years, new treatment approaches have led to higher rates of complete tumor eradication combined with organ-preservation strategies. However, better tools are still needed to personalize therapy for the individual patient. In this prospective observational study, we analyzed colon-derived cell-free (cf)DNA (c-cfDNA) using a tissue-specific DNA methylation signature, and its association with therapy outcomes. Analyzing plasma samples (n = 303) collected during nCRT from 37 patients with locally advanced rectal cancer (LARC), we identified colon-specific methylation markers that discriminated healthy individuals from patients with untreated LARC (area under the curve, 0.81; 95% confidence interval, 0.70-0.92; P < .0001). Baseline c-cfDNA predicted tumor response, with increased levels linked to larger residual cancer. c-cfDNA measured after the first week of therapy identified patients with maximal response and complete cancer eradication, who had significantly lower c-cfDNA compared with those who had residual disease (8.6 vs 57.7 average copies/ml, respectively; P = .013). Increased c-cfDNA after 1 week of therapy was also associated with disease recurrence. Methylation-based liquid biopsy can predict nCRT outcomes and facilitate patient selection for escalation and de-escalation strategies.
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Ácidos Nucleicos Livres , Neoplasias Retais , Humanos , Ácidos Nucleicos Livres/genética , Recidiva Local de Neoplasia , Quimiorradioterapia , Neoplasias Retais/genética , Neoplasias Retais/terapia , Neoplasias Retais/patologia , Reto/patologia , Terapia Neoadjuvante , Resultado do Tratamento , Estudos RetrospectivosRESUMO
The use of cardiopulmonary bypass (CPB) is thought to cause delayed cardiac damage. DNA methylation-based liquid biopsies are novel biomarkers for monitoring acute cardiac cell death. We assessed cell-free DNA molecules as markers for cardiac damage after open-heart surgery. Novel cardiomyocyte-specific DNA methylation markers were applied to measure cardiac cfDNA in the plasma of 42 infants who underwent open-heart surgery. Cardiac cfDNA was elevated following surgery, reflecting direct surgery-related tissue damage, and declined thereafter in most patients. The concentration of cardiac cfDNA post-surgery correlated with the duration of CPB and aortic cross clamping. Strikingly, cardiac cfDNA at 6 h predicted duration of mechanical ventilation and maximal vasoactive-inotropic score better than did maximal troponin levels. Cardiac cfDNA reveals heart damage associated with CPB, and can be used to monitor cardiac cell death, to predict clinical outcome of surgery and to assess performance of cardioprotective interventions.
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Procedimentos Cirúrgicos Cardíacos , Ácidos Nucleicos Livres , Lactente , Humanos , Biomarcadores , Morte Celular , Metilação de DNARESUMO
The Oxford Nanopore (ONT) platform provides portable and rapid genome sequencing, and its ability to natively profile DNA methylation without complex sample processing is attractive for point-of-care real-time sequencing. We recently demonstrated ONT shallow whole-genome sequencing to detect copy number alterations (CNAs) from the circulating tumor DNA (ctDNA) of cancer patients. Here, we show that cell type and cancer-specific methylation changes can also be detected, as well as cancer-associated fragmentation signatures. This feasibility study suggests that ONT shallow WGS could be a powerful tool for liquid biopsy.
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Ácidos Nucleicos Livres , DNA Tumoral Circulante , Sequenciamento por Nanoporos , Neoplasias , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/genéticaRESUMO
Schizophrenia is a common, severe, and debilitating psychiatric disorder. Despite extensive research there is as yet no biological marker that can aid in its diagnosis and course prediction. This precludes early detection and intervention. Imaging studies suggest brain volume loss around the onset and over the first few years of schizophrenia, and apoptosis has been proposed as the underlying mechanism. Cell-free DNA (cfDNA) fragments are released into the bloodstream following cell death. Tissue-specific methylation patterns allow the identification of the tissue origins of cfDNA. We developed a cocktail of brain-specific DNA methylation markers, and used it to assess the presence of brain-derived cfDNA in the plasma of patients with a first psychotic episode. We detected significantly elevated neuron- (p=0.0013), astrocyte- (p=0.0016), oligodendrocyte- (p=0.0129), and whole brain-derived (p=0.0012) cfDNA in the plasma of patients during their first psychotic episode (n=29), compared with healthy controls (n=31). Increased cfDNA levels were not correlated with psychotropic medications use. Area under the curve (AUC) was 0.77, with 65% sensitivity at 90% specificity in patients with a psychotic episode. Potential interpretations of these findings include increased brain cell death, disruption of the blood-brain barrier, or a defect in clearance of material from dying brain cells. Brain-specific cfDNA methylation markers can potentially assist early detection and monitoring of schizophrenia and thus allow early intervention and adequate therapy.
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Ácidos Nucleicos Livres , Transtornos Psicóticos , Biomarcadores Tumorais/genética , Encéfalo , Ácidos Nucleicos Livres/genética , Metilação de DNA , Marcadores Genéticos , Humanos , Transtornos Psicóticos/genéticaRESUMO
BACKGROUND: Much remains unknown regarding the response of the immune system to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccination. METHODS: We employed circulating cell-free DNA (cfDNA) to assess the turnover of specific immune cell types following administration of the Pfizer/BioNTech vaccine. FINDINGS: The levels of B cell cfDNA after the primary dose correlated with development of neutralizing antibodies and memory B cells after the booster, revealing a link between early B cell turnover-potentially reflecting affinity maturation-and later development of effective humoral response. We also observed co-elevation of B cell, T cell, and monocyte cfDNA after the booster, underscoring the involvement of innate immune cell turnover in the development of humoral and cellular adaptive immunity. Actual cell counts remained largely stable following vaccination, other than a previously demonstrated temporary reduction in neutrophil and lymphocyte counts. CONCLUSIONS: Immune cfDNA dynamics reveal the crucial role of the primary SARS-CoV-2 vaccine in shaping responses of the immune system following the booster vaccine. FUNDING: This work was supported by a generous gift from Shlomo Kramer. Supported by grants from Human Islet Research Network (HIRN UC4DK116274 and UC4DK104216 to R.S. and Y.D.), Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine, The Alex U Soyka Pancreatic Cancer Fund, The Israel Science Foundation, the Waldholtz/Pakula family, the Robert M. and Marilyn Sternberg Family Charitable Foundation, the Helmsley Charitable Trust, Grail, and the DON Foundation (to Y.D.). Y.D. holds the Walter and Greta Stiel Chair and Research Grant in Heart Studies. I.F.-F. received a fellowship from the Glassman Hebrew University Diabetes Center.
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Vacina BNT162 , COVID-19 , Ácidos Nucleicos Livres , SARS-CoV-2 , Adulto , Idoso , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Vacina BNT162/administração & dosagem , COVID-19/imunologia , COVID-19/prevenção & controle , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/imunologia , Feminino , Humanos , Imunização Secundária , Masculino , Células B de Memória/imunologia , Células B de Memória/metabolismo , Pessoa de Meia-Idade , SARS-CoV-2/imunologia , Adulto JovemRESUMO
BACKGROUND: Circulating biomarkers for lung damage are lacking. Lung epithelium-specific DNA methylation patterns can potentially report the presence of lung-derived cell-free DNA (cfDNA) in blood, as an indication of lung cell death. METHODS: We sorted human lung alveolar and bronchial epithelial cells from surgical specimens, and obtained their methylomes using whole-genome bisulfite sequencing. We developed a PCR sequencing assay determining the methylation status of 17 loci with lung-specific methylation patterns, and used it to assess lung-derived cfDNA in the plasma of healthy volunteers and patients with lung disease. RESULTS: Loci that are uniquely unmethylated in alveolar or bronchial epithelial cells are enriched for enhancers controlling lung-specific genes. Methylation markers extracted from these methylomes revealed that normal lung cell turnover probably releases cfDNA into the air spaces, rather than to blood. People with advanced lung cancer show a massive elevation of lung cfDNA concentration in blood. Among individuals undergoing bronchoscopy, lung-derived cfDNA is observed in the plasma of those later diagnosed with lung cancer, and to a lesser extent in those diagnosed with other lung diseases. Lung cfDNA is also elevated in patients with acute exacerbation of COPD compared with patients with stable disease, and is associated with future exacerbation and mortality in these patients. CONCLUSIONS: Universal cfDNA methylation markers of normal lung epithelium allow for mutation-independent, sensitive and specific detection of lung-derived cfDNA, reporting on ongoing lung injury. Such markers can find broad utility in the study of normal and pathologic human lung dynamics.
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Ácidos Nucleicos Livres , Neoplasias Pulmonares , Humanos , Metilação de DNA , Ácidos Nucleicos Livres/genética , Biópsia Líquida , Biomarcadores , Epitélio , Pulmão , Neoplasias Pulmonares/genética , Biomarcadores Tumorais/genéticaRESUMO
Cancer inflicts damage to surrounding normal tissues, which can culminate in fatal organ failure. Here, we demonstrate that cell death in organs affected by cancer can be detected by tissue-specific methylation patterns of circulating cell-free DNA (cfDNA). We detected elevated levels of hepatocyte-derived cfDNA in the plasma of patients with liver metastases originating from different primary tumors, compared with cancer patients without liver metastases. In addition, patients with localized pancreatic or colon cancer showed elevated hepatocyte cfDNA, suggesting liver damage inflicted by micrometastatic disease, by primary pancreatic tumor pressing the bile duct, or by a systemic response to the primary tumor. We also identified elevated neuron-, oligodendrocyte-, and astrocyte-derived cfDNA in a subpopulation of patients with brain metastases compared with cancer patients without brain metastasis. Cell type-specific cfDNA methylation markers enabled the identification of collateral tissue damage in cancer, revealing the presence of metastases in specific locations and potentially assisting in early cancer detection.