Elevations in Circulating Methylated and Unmethylated Preproinsulin DNA in New-Onset Type 1 Diabetes

Fisher, Marisa M.; Watkins, Renecia A.; Blum, Janice S.; Evans‐Molina, Carmella; Chalasani, Naga; DiMeglio, Linda A.; Mather, Kieren J.; Tersey, Sarah A.; Mirmira, Raghavendra G.

doi:10.2337/db15-0430

Cited by 81 publications

(119 citation statements)

References 16 publications

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“…Previously published cfDNA assays that detect β-cell death in T1D were limited by high background signal in healthy control subjects (21,22,24,25). We interpreted this signal as being caused by chance demethylation of CpGs in cfDNA from non-β-cell tissues.…”

Section: Discussionmentioning

confidence: 95%

“…Recent studies have identified unmethylated INS promoter DNA in the circulation of patients with recently diagnosed T1D and in islet-graft recipients, likely reflecting both autoimmune and alloimmune destruction of β cells (21)(22)(23)(24)(25). However, published data suggest that the analytic approaches used were not sufficiently specific to differentiate robustly between β-cell-and non-β-cellderived cfDNA.…”

Section: Significancementioning

confidence: 99%

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Identification of tissue-specific cell death using methylation patterns of circulating DNA

Lehmann-Werman¹,

Neiman²,

Zemmour³

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

523

538

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Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic β-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

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Section: Discussionmentioning

confidence: 95%

Section: Significancementioning

confidence: 99%

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Lehmann-Werman¹,

Neiman²,

Zemmour³

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

523

538

View full text Add to dashboard Cite

show abstract

“…4D). To assess β-cell apoptosis in vivo further, droplet digital PCR was used to measure circulating levels of unmethylated cell-free insulin 2 (Ins2) DNA in serum (41)(42)(43). Importantly, Atf5…”

Section: Atf5 Regulates the Susceptibility Of β Cells To Stress-inducedmentioning

confidence: 99%

ATF5 regulates β-cell survival during stress

Juliana

Yang

Rozo

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The stress response and cell survival are necessary for normal pancreatic β-cell function, glucose homeostasis, and prevention of diabetes. The homeodomain transcription factor and human diabetes gene pancreas/duodenum homeobox protein 1 (Pdx1) regulates β-cell survival and endoplasmic reticulum stress susceptibility, in part through direct regulation of activating transcription factor 4 (Atf4). Here we show that Atf5, a close but less-studied relative of Atf4, is also a target of Pdx1 and is critical for β-cell survival under stress conditions. Pdx1 deficiency led to decreased Atf5 transcript, and primary islet ChIP-sequencing localized PDX1 to the Atf5 promoter, implicating Atf5 as a PDX1 target. Atf5 expression was stress inducible and enriched in β cells. Importantly, Atf5 deficiency decreased survival under stress conditions. Loss-of-function and chromatin occupancy experiments positioned Atf5 downstream of and parallel to Atf4 in the regulation of eIF4E-binding protein 1 (4ebp1), a mammalian target of rapamycin (mTOR) pathway component that inhibits protein translation. Accordingly, Atf5 deficiency attenuated stress suppression of global translation, likely enhancing the susceptibility of β cells to stress-induced apoptosis. Thus, we identify ATF5 as a member of the transcriptional network governing pancreatic β-cell survival during stress.educed pancreatic β-cell number and function characterize all forms of diabetes. Insulin-secreting β cells are notoriously susceptible to stress, including endoplasmic reticulum (ER), cytokine, and oxidative stress (1-4). Thus, understanding apoptotic cell-fate decisions during stress could provide new targets that could be exploited for the prevention or amelioration of diabetes.In secretory cells such as the β cell, the unfolded protein response (UPR) and regulation of translation, particularly in response to stress, are key factors in maintaining cellular homeostasis, as clearly demonstrated in mouse models with deficiencies of critical regulators such as protein kinase R-like ER kinase (PERK) and EIF2α (5-7). In humans, PERK mutation causes Wolcott-Rallison syndrome, a rare autosomal recessive disorder characterized by permanent neonatal diabetes (8, 9). Downstream of PERK, the basic leucine zipper (bZIP) transcription factor activating transcription factor 4 (ATF4) regulates the expression of 4ebp1, a member of the eukaryotic translation initiation factor 4E (eIF4E)-binding protein family (4EBPs). 4EBP1 is the most abundant mammalian isoform in the pancreas (10) and functions as an inhibitor of translation initiation by binding the capbinding protein eIF4E, thereby preventing formation of the eIF4F translational initiation complex (11,12). Expression of 4EBP1 is induced by stress, and 4ebp1 deficiency results in deregulated translational control and increased susceptibility to ER stressmediated apoptosis in β cells (13).We previously demonstrated that the homeodomain transcription factor and human diabetes gene pancreas/duodenum homeobox protein 1 (Pdx1) regulates p...

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“…ddPCR to verify mutation was done as described previously 21 . Samples were analyzed using a dual fluorescent probe-based multiplex assay.…”

Section: Methodsmentioning

confidence: 99%

A system for detecting high impact-low frequency mutations in primary tumors and metastases

et al. 2017

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Tumor complexity and intratumor heterogeneity contribute to subclonal diversity. Despite advances in next-generation sequencing (NGS) and bioinformatics, detecting rare mutations in primary tumors and metastases contributing to subclonal diversity is a challenge for precision genomics. Here, in order to identify rare mutations, we adapted a recently described epithelial reprograming assay for short-term propagation of epithelial cells from primary and metastatic tumors. Using this approach, we expanded minor clones and obtained epithelial cell-specific DNA/RNA for quantitative NGS analysis. Comparative Ampliseq Comprehensive Cancer Panel sequence analyses were performed on DNA from unprocessed breast tumor and tumor cells propagated from the same tumor. We identified previously uncharacterized mutations present only in the cultured tumor cells, a subset of which has been reported in brain metastatic but not primary breast tumors. In addition, whole-genome sequencing identified mutations enriched in liver metastases of various cancers, including Notch pathway mutations/chromosomal inversions in 5/5 liver metastases, irrespective of cancer types. Mutations/rearrangements in FHIT, involved in purine metabolism, were detected in 4/5 liver metastases, and the same four liver metastases shared mutations in 32 genes, including mutations of different HLA-DR family members affecting OX40 signaling pathway, which could impact the immune response to metastatic cells. Pathway analyses of all mutated genes in liver metastases showed aberrant tumor necrosis factor and transforming growth factor signaling in metastatic cells. Epigenetic regulators including KMT2C/MLL3 and ARID1B, which are mutated in >50% of hepatocellular carcinomas, were also mutated in liver metastases. Thus, irrespective of cancer types, organ-specific metastases may share common genomic aberrations. Since recent studies show independent evolution of primary tumors and metastases and in most cases mutation burden is higher in metastases than primary tumors, the method described here may allow early detection of subclonal somatic alterations associated with metastatic progression and potentially identify therapeutically actionable, metastasis-specific genomic aberrations.

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Elevations in Circulating Methylated and Unmethylated Preproinsulin DNA in New-Onset Type 1 Diabetes

Cited by 81 publications

References 16 publications

Identification of tissue-specific cell death using methylation patterns of circulating DNA

Identification of tissue-specific cell death using methylation patterns of circulating DNA

ATF5 regulates β-cell survival during stress

A system for detecting high impact-low frequency mutations in primary tumors and metastases

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