circulating cell free dna
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eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ilana Fox-Fisher ◽  
Sheina Piyanzin ◽  
Bracha Lea Ochana ◽  
Agnes Klochendler ◽  
Judith Magenheim ◽  
...  

Blood cell counts often fail to report on immune processes occurring in remote tissues. Here we use immune cell type-specific methylation patterns in circulating cell-free DNA (cfDNA) for studying human immune cell dynamics. We characterized cfDNA released from specific immune cell types in healthy individuals (N=242), cross sectionally and longitudinally. Immune cfDNA levels had no individual steady state as opposed to blood cell counts, suggesting that cfDNA concentration reflects adjustment of cell survival to maintain homeostatic cell numbers. We also observed selective elevation of immune-derived cfDNA upon perturbations of immune homeostasis. Following influenza vaccination (N=92), B-cell-derived cfDNA levels increased prior to elevated B-cell counts and predicted efficacy of antibody production. Patients with Eosinophilic Esophagitis (N=21) and B-cell lymphoma (N=27) showed selective elevation of eosinophil and B-cell cfDNA respectively, which were undetectable by cell counts in blood. Immune-derived cfDNA provides a novel biomarker for monitoring immune responses to physiological and pathological processes that are not accessible using conventional methods.


2021 ◽  
pp. 1-13
Author(s):  
Lei Chen ◽  
Qianqian Shen ◽  
Shunliang Xu ◽  
Hongzhuan Yu ◽  
Shengjie Pei ◽  
...  

Background: 5-Hydroxymethylcytosine (5hmC) is an epigenetic DNA modification that is highly abundant in central nervous system. It has been reported that DNA 5hmC dysregulation play a critical role in Alzheimer’s disease (AD) pathology. Changes in 5hmC signatures can be detected in circulating cell-free DNA (cfDNA), which has shown potential as a non-invasive liquid biopsy material. Objective: However, the genome-wide profiling of 5hmC in cfDNA and its potential for the diagnosis of AD has not been reported to date. Methods: We carried out a case-control study and used a genome-wide chemical capture followed by high-throughput sequencing to detect the genome-wide profiles of 5hmC in human cfDNA and identified differentially hydroxymethylated regions (DhMRs) in late-onset AD patients and the control. Results: We discovered significant differences of 5hmC enrichment in gene bodies which were linked to multiple AD pathogenesis-associated signaling pathways in AD patients compared with cognitively normal controls, indicating they can be well distinguished from normal controls by DhMRs in cfDNA. Specially, we identified 7 distinct genes (RABEP1, CPNE4, DNAJC15, REEP3, ROR1, CAMK1D, and RBFOX1) with predicting diagnostic potential based on their significant correlations with MMSE and MoCA scores of subjects. Conclusion: The present results suggest that 5hmC markers derived from plasma cfDNA can served as an effective, minimally invasive biomarkers for clinical auxiliary diagnosis of late-onset AD.


eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Nicole Laurencia Yuwono ◽  
Kristina Warton ◽  
Caroline Elizabeth Ford

Research and clinical use of circulating cell-free DNA (cirDNA) is expanding rapidly; however, there remain large gaps in our understanding of the influence of lifestyle and biological factors on the amount of cirDNA present in blood. Here, we review 66 individual studies of cirDNA levels and lifestyle and biological factors, including exercise (acute and chronic), alcohol consumption, occupational hazard exposure, smoking, body mass index, menstruation, hypertension, circadian rhythm, stress, biological sex and age. Despite technical and methodological inconsistences across studies, we identify acute exercise as a significant influence on cirDNA levels. Given the large increase in cirDNA induced by acute exercise, we recommend that controlling for physical activity prior to blood collection is routinely incorporated into study design when total cirDNA levels are of interest. We also highlight appropriate selection and complete reporting of laboratory protocols as important for improving the reproducibility cirDNA studies and ability to critically evaluate the results.


2021 ◽  
Author(s):  
Rafael Ricardo de Castro Cuadrat ◽  
Adelheid Kratzer ◽  
Hector Giral Arnal ◽  
Katarzyna Wreczycka ◽  
Alexander Blume ◽  
...  

Acute coronary syndromes (ACS) remain a major cause of worldwide mortality. ACS diagnosis is done by a combination of factors, such as electrocardiogram and plasma biomarkers. These biomarkers, however, lack the power to accurately stratify patients into different risk groups. Instead, we used changes in the circulating cell-free DNA (ccfDNA) methylation profiles to estimate the extent of heart injury and the severity of ACS. Our approach relies on the fact that dying cells in acutely damaged tissue release DNA into the blood, causing an increase in the ccfDNA. In addition, each cell type has a distinct DNA methylation profile. We leverage cell type/state specificity of DNA methylation to deconvolute the cell types of origin for ccfDNA and also find DNA methylation-based biomarkers that stratify patient cohorts. The cohorts consisted of healthy subjects, and patients from three ACS conditions: ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI) and unstable angina (UA). We have used two cohorts of patients - discovery, and validation, both consisting of the same conditions . We have sequenced the ccfDNA from the discovery cohort using Whole Bisulfite Genome Sequencing (WBGS), to obtain an unbiased overview of plasma DNA methylation profiles. We have found a total of 1,614 differential methylated regions (DMRs) in the three ACS groups. Many of the regions are associated with genes involved in cardiovascular conditions and inflammation. Using linear models we were able to narrow down to 254 DMRs significantly associated with ACS severity. The reduced list of DMRs enabled a more accurate stratification of ACS patients. The predictive power of the DMRs was validated in the confirmation cohort using targeted methylation sequencing of the validation cohort. Measuring methylation on ccfDNA showed promise as a method for estimating the level of heart injury during an acute coronary event, and accurate patient risk stratification. The method is however not limited to acute events, and can be extended to other heart related diseases. It can be used for estimating the status of the disease in patients with chronic states, such as heart failure and coronary artery disease.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4931-4931
Author(s):  
Karolliny Silva de Oliveira ◽  
Hebert Fabricio Culler ◽  
Débora Levy ◽  
José Roberto Assis Filho ◽  
Daniel Silva Nogueira ◽  
...  

Abstract Introduction: Isolation of circulating cell-free DNA (ccfDNA) and circulating tumor DNA (ctDNA) are useful methodologies for studies of neoplastic genetic material and can provide information about cancer heterogeneity, prognosis and minimal residual disease (MRD) [1,2]. Therefore, the recovery of ccfDNA in sufficient quantity and adequate purity is crucial to provide accurate and reproducible results. In this study, we investigate the efficiency of four different commercial kits to recover short fragments of DNA compatible with the ccfDNA. Methods: Ten mL of total peripheral blood from seven patients with Diffuse Large B-Cell Lymphoma (DLBCL) were collected in EDTA and were immediately processed. Plasma was obtained by centrifugation at 1.900 x g for 10 min at 4 ºC, followed by other centrifugation at 20.000 x g for 10 min at 4 ºC to remove cellular debris. Aliquots of 1.0 mL of plasma were stored at -80 ºC until ccfDNA extraction. The ccfDNA extraction was performed using the same samples by the following kits: QIAamp MinElute ccfDNA kit (CFDNA - Qiagen, Hilden, Germany), QIAamp DNA Mini Kit (QBLOOD - Qiagen, Hilden, Germany), Illustra Blood GenomicPrep Mini Spin Kit (GE - GE Healthcare Bio-Sciences Corp, UK) and ccfDNA isolation kit Quick-ccfDNA Serum & Plasma Kit (ZYMO - Zymo Research, Irvine, USA) [Table 1] according to the manufacturer's recommendations. The ccfDNA obtained was eluted in 30 µL of elution buffer for the subsequent experiments. The measurement of the ccfDNA obtained for each kit was performed in a Qubit 2.0 fluorometer using Qubit dsDNA HS assay kit (Thermo Fischer Scientific, Waltham, USA). The analysis of integrity, purity and the size of the fragments of DNA were performed in the Agilent 2100 Bioanalyzer (Agilent Technologies, Germany) equipment using the High Sensitivity DNA kit (Agilent Technologies, Germany). Results: The results obtained for each commercial kit in the Qubit-analysis were: CFDNA (0.27 ng/uL), QBLOOD (0.26 ng/uL), ZYMO (0.03 ng/uL) and GE (0.02 ng/uL) [Figure 1]. The Bionalyzer eletrophoresis showed that CFDNA kit was able to recover two different fragments sizes of ccfDNA one of 360-380 bp (0.114 ng/uL) and other of 160-180 bp (1.808 ng/uL). The DNA recovered with QBLOOD revealed sizes of 360-380 bp (0.031 ng/uL) and 160-180 bp (0.079 ng/uL). Both kits, GE and ZYMO were not able to isolate fragments of DNA smaller than 10.380 pb . Conclusion: We demonstrated that QIAamp MinElute ccfDNA kit (CFDNA) was capable to recovery high quantity of small fragments of DNA compatible with ccfDNA. This result supports the use of protocols based in magnetic beads in experiments working with ccfDNA. Additionally, we highlight the importance to verify the quality and not only the quantity of the DNA extracted to confirm the presence of ccfDNA. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.


2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi14-vi14
Author(s):  
Jacob Till ◽  
Aseel Abdalla ◽  
Zhuoyang Wang ◽  
Wanding Zhou ◽  
S Ali Nabavizadeh ◽  
...  

Abstract We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.


Author(s):  
Anais Alonso ◽  
Nicole Laurencia Yuwono ◽  
Sahar Houshdaran ◽  
Jason Abbott ◽  
Rachael Rodgers ◽  
...  

2021 ◽  
Author(s):  
Gabrielle Smith ◽  
Juliane Lippert ◽  
Barbara Altieri ◽  
Yasir Elhassan ◽  
Laura Landwehr ◽  
...  

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