DNA Methylation-Based Assessment of Cell Composition in Human Pancreas and Islets.

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.

Chronic graft-versus-host disease detected by tissue-specific cell-free DNA methylation biomarkers.

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).

Protein biomarkers and alternatively methylated cell-free DNA detect early stage pancreatic cancer.

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.

Beta-cell death and dysfunction drives hyperglycaemia in organ donors.

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.

The DNA methylome of human vascular endothelium and its use in liquid biopsies.

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.

A universal cell-free DNA approach for response prediction to preoperative chemoradiation in rectal cancer.

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.

B cell-derived cfDNA after primary BNT162b2 mRNA vaccination anticipates memory B cells and SARS-CoV-2 neutralizing antibodies.

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.

Elevated brain-derived cell-free DNA among patients with first psychotic episode - a proof-of-concept study.

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.

Universal lung epithelium DNA methylation markers for detection of lung damage in liquid biopsies.

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.

Liquid biopsy reveals collateral tissue damage in cancer.

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.

ChIP-seq of plasma cell-free nucleosomes identifies gene expression programs of the cells of origin.

Cell-free DNA (cfDNA) in human plasma provides access to molecular information about the pathological processes in the organs or tumors from which it originates. These DNA fragments are derived from fragmented chromatin in dying cells and retain some of the cell-of-origin histone modifications. In this study, we applied chromatin immunoprecipitation of cell-free nucleosomes carrying active chromatin modifications followed by sequencing (cfChIP-seq) to 268 human samples. In healthy donors, we identified bone marrow megakaryocytes, but not erythroblasts, as major contributors to the cfDNA pool. In patients with a range of liver diseases, we showed that we can identify pathology-related changes in hepatocyte transcriptional programs. In patients with metastatic colorectal carcinoma, we detected clinically relevant and patient-specific information, including transcriptionally active human epidermal growth factor receptor 2 (HER2) amplifications. Altogether, cfChIP-seq, using low sequencing depth, provides systemic and genome-wide information and can inform diagnosis and facilitate interrogation of physiological and pathological processes using blood samples.

Multiplexing DNA methylation markers to detect circulating cell-free DNA derived from human pancreatic ß cells.

It has been proposed that unmethylated insulin promoter fragments in plasma derive exclusively from ß cells, reflect their recent demise, and can be used to assess ß cell damage in type 1 diabetes. Herein we describe an ultrasensitive assay for detection of a ß cell-specific DNA methylation signature, by simultaneous assessment of 6 DNA methylation markers, that identifies ß cell DNA in mixtures containing as little as 0.03% ß cell DNA (less than 1 ß cell genome equivalent). Based on this assay, plasma from nondiabetic individuals (N = 218, aged 4-78 years) contained on average only 1 ß cell genome equivalent/mL. As expected, cell-free DNA (cfDNA) from ß cells was significantly elevated in islet transplant recipients shortly after transplantation. We also detected ß cell cfDNA in a patient with KATP congenital hyperinsulinism, in which substantial ß cell turnover is thought to occur. Strikingly, in contrast to previous reports, we observed no elevation of ß cell-derived cfDNA in autoantibody-positive subjects at risk for type 1 diabetes (N = 32), individuals with recent-onset type 1 diabetes (<4 months, N = 92), or those with long-standing disease (>4 months, N = 38). We discuss the utility of sensitive ß cell cfDNA analysis and potential explanations for the lack of a ß cell cfDNA signal in type 1 diabetes.

Circulating Unmethylated Insulin DNA As a Biomarker of Human Beta Cell Death: A Multi-laboratory Assay Comparison.

CONTEXT: There is an unmet need for biomarkers of pancreatic beta-cell death to improve early diagnosis of type 1 diabetes, enroll subjects into clinical trials, and assess treatment response. To address this need, several groups developed assays measuring insulin deoxyribonucleic acid (DNA) with unmethylated CpG sites in cell-free DNA. Unmethylated insulin DNA should be derived predominantly from beta-cells and indicate ongoing beta-cell death. OBJECTIVE: To assess the performance of three unmethylated insulin DNA assays. DESIGN AND PARTICIPANTS: Plasma or serum samples from 13 subjects undergoing total pancreatectomy and islet autotransplantation were coded and provided to investigators to measure unmethylated insulin DNA. Samples included a negative control taken post-pancreatectomy but pretransplant, and a positive control taken immediately following islet infusion. We assessed technical reproducibility, linearity, and persistence of detection of unmethylated insulin DNA for each assay. RESULTS: All assays discriminated between the negative sample and samples taken directly from the islet transplant bag; 2 of 3 discriminated negative samples from those taken immediately after islet infusion. When high levels of unmethylated insulin DNA were present, technical reproducibility was generally good for all assays. CONCLUSIONS: The measurement of beta cell cell-free DNA, including insulin, is a promising approach, warranting further testing and development in those with or at-risk for type 1 diabetes, as well as in other settings where understanding the frequency or kinetics of beta cell death could be useful.

Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease.

Methylation patterns of circulating cell-free DNA (cfDNA) contain rich information about recent cell death events in the body. Here, we present an approach for unbiased determination of the tissue origins of cfDNA, using a reference methylation atlas of 25 human tissues and cell types. The method is validated using in silico simulations as well as in vitro mixes of DNA from different tissue sources at known proportions. We show that plasma cfDNA of healthy donors originates from white blood cells (55%), erythrocyte progenitors (30%), vascular endothelial cells (10%) and hepatocytes (1%). Deconvolution of cfDNA from patients reveals tissue contributions that agree with clinical findings in sepsis, islet transplantation, cancer of the colon, lung, breast and prostate, and cancer of unknown primary. We propose a procedure which can be easily adapted to study the cellular contributors to cfDNA in many settings, opening a broad window into healthy and pathologic human tissue dynamics.

Monitoring liver damage using hepatocyte-specific methylation markers in cell-free circulating DNA.

Liver damage is typically inferred from serum measurements of cytoplasmic liver enzymes. DNA molecules released from dying hepatocytes are an alternative biomarker, unexplored so far, potentially allowing for quantitative assessment of liver cell death. Here we describe a method for detecting acute hepatocyte death, based on quantification of circulating, cell-free DNA (cfDNA) fragments carrying hepatocyte-specific methylation patterns. We identified 3 genomic loci that are unmethylated specifically in hepatocytes, and used bisulfite conversion, PCR, and massively parallel sequencing to quantify the concentration of hepatocyte-derived DNA in mixed samples. Healthy donors had, on average, 30 hepatocyte genomes/ml plasma, reflective of basal cell turnover in the liver. We identified elevations of hepatocyte cfDNA in patients shortly after liver transplantation, during acute rejection of an established liver transplant, and also in healthy individuals after partial hepatectomy. Furthermore, patients with sepsis had high levels of hepatocyte cfDNA, which correlated with levels of liver enzymes aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Duchenne muscular dystrophy patients, in which elevated AST and ALT derive from damaged muscle rather than liver, did not have elevated hepatocyte cfDNA. We conclude that measurements of hepatocyte-derived cfDNA can provide specific and sensitive information on hepatocyte death, for monitoring human liver dynamics, disease, and toxicity.

Non-invasive detection of human cardiomyocyte death using methylation patterns of circulating DNA.

Detection of cardiomyocyte death is crucial for the diagnosis and treatment of heart disease. Here we use comparative methylome analysis to identify genomic loci that are unmethylated specifically in cardiomyocytes, and develop these as biomarkers to quantify cardiomyocyte DNA in circulating cell-free DNA (cfDNA) derived from dying cells. Plasma of healthy individuals contains essentially no cardiomyocyte cfDNA, consistent with minimal cardiac turnover. Patients with acute ST-elevation myocardial infarction show a robust cardiac cfDNA signal that correlates with levels of troponin and creatine phosphokinase (CPK), including the expected elevation-decay dynamics following coronary angioplasty. Patients with sepsis have high cardiac cfDNA concentrations that strongly predict mortality, suggesting a major role of cardiomyocyte death in mortality from sepsis. A cfDNA biomarker for cardiomyocyte death may find utility in diagnosis and monitoring of cardiac pathologies and in the study of normal human cardiac physiology and development.

Beta Cell Death by Cell-free DNA and Outcome After Clinical Islet Transplantation.

BACKGROUND: Optimizing engraftment and early survival after clinical islet transplantation is critical to long-term function, but there are no reliable, quantifiable measures to assess beta cell death. Circulating cell-free DNA (cfDNA) derived from beta cells has been identified as a novel biomarker to detect cell loss and was recently validated in new-onset type 1 diabetes and in islet transplant patients. METHODS: Herein we report beta cell cfDNA measurements after allotransplantation in 37 subjects and the correlation with clinical outcomes. RESULTS: A distinctive peak of cfDNA was observed 1 hour after transplantation in 31 (83.8%) of 37 subjects. The presence and magnitude of this signal did not correlate with transplant outcome. The 1-hour signal represents dead beta cells carried over into the recipient after islet isolation and culture, combined with acute cell death post infusion. Beta cell cfDNA was also detected 24 hours posttransplant (8/37 subjects, 21.6%). This signal was associated with higher 1-month insulin requirements (P = 0.04), lower 1-month stimulated C-peptide levels (P = 0.01), and overall worse 3-month engraftment, by insulin independence (receiver operating characteristic-area under the curve = 0.70, P = 0.03) and beta 2 score (receiver operating characteristic-area under the curve = 0.77, P = 0.006). CONCLUSIONS: cfDNA-based estimation of beta cell death 24 hours after islet allotransplantation correlates with clinical outcome and could predict early engraftment.

Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type-specific methylation in enhancers.

DNA methylation at promoters is an important determinant of gene expression. Earlier studies suggested that the insulin gene promoter is uniquely unmethylated in insulin-expressing pancreatic ß-cells, providing a classic example of this paradigm. Here we show that islet cells expressing insulin, glucagon, or somatostatin share a lack of methylation at the promoters of the insulin and glucagon genes. This is achieved by rapid demethylation of the insulin and glucagon gene promoters during differentiation of Neurogenin3+ embryonic endocrine progenitors, regardless of the specific endocrine cell-type chosen. Similar methylation dynamics were observed in transgenic mice containing a human insulin promoter fragment, pointing to the responsible cis element. Whole-methylome comparison of human α- and ß-cells revealed generality of the findings: genes active in one cell type and silent in the other tend to share demethylated promoters, while methylation differences between α- and ß-cells are concentrated in enhancers. These findings suggest an epigenetic basis for the observed plastic identity of islet cell types, and have implications for ß-cell reprogramming in diabetes and diagnosis of ß-cell death using methylation patterns of circulating DNA.