Islet β cell death precedes development of type 1 diabetes, and detecting this process may allow for early therapeutic intervention. Here, we provide a detailed description of how to measure differentially methylated INS DNA species in human serum as a biomarker of β cell death.
The death of islet β cells is thought to underlie the pathogenesis of virtually all forms of diabetes and to precede the development of frank hyperglycemia, especially in type 1 diabetes. The development of sensitive and reliable biomarkers of β cell death may allow for early therapeutic intervention to prevent or delay the development of diabetes. Recently, several groups including our own have reported that cell-free, differentially methylated DNA encoding preproinsulin (INS) in the circulation is correlated to β cell death in pre-type 1 diabetes and new-onset type 1 diabetes. Here, we present a step-by-step protocol using digital PCR for the measurement of cell-free INS DNA that is differentially methylated at cytosine at position -69 bp (relative to the transcriptional start site). We demonstrate that the assay can distinguish between methylated and unmethylated cytosine at position -69 bp, is linear across several orders of magnitude, provides absolute quantitation of DNA copy numbers, and can be applied to samples of human serum from individuals with new-onset type 1 diabetes and disease-free controls. The protocol described here can be adapted to any DNA species for which detection of differentially methylated cytosines is desired, whether from circulation or from isolated cells and tissues, and can provide absolute quantitation of DNA fragments.
Type 1 diabetes (T1D) is an autoimmune disease that is characterized by the destruction of insulin-producing islet β cells by autoreactive T cells1. The diagnosis of T1D is typically made upon measurement of hyperglycemia (blood glucose > 200 mg/dl) in a lean, young individual, who might present with ketoacidosis as evidence of insulin deficiency. At the time of diagnosis of T1D, there is evidence for substantial loss of β cell function and mass (from 50 – 90%)2. In clinical studies, several immune modulatory drugs that were instituted at the time of diagnosis resulted in the stabilization of β cell function (and presumably mass), but none have resulted in clinical remission of disease, a finding that has raised the call for the development of biomarkers for earlier detection of the disease and for the longitudinal tracking of effectiveness of combination therapies3,4. Efforts by international consortia, such as the Human Islet Research Network Consortium at the National Institutes of Heath5, have emphasized the need to develop biomarkers that focus on β cell stress and death in T1D.
In line with these efforts, our group and others have recently developed biomarker assays that measure circulating, epigenetically modified DNA fragments that arise primarily from dying β cells6–9. In all of the published assays to date, the focus has been on the quantitation of the human gene encoding preproinsulin (INS), which demonstrates greater degrees of unmethylated CpG sites in the coding and promoter regions compared to other cell types. The liberation of unmethylated INS DNA fragments was hypothesized as arising primarily from dying (necrotic, apoptotic) β cells. Our recent studies showed that in youth, elevations in both unmethylated and methylated INS DNA at position -69 bp (relative to the transcriptional start site) were observed in new-onset T1D, and together served as specific biomarkers for this population6. These biomarker assays involve the isolation of cell-free DNA from serum or plasma using commercial spin kits, followed by a bisulfite conversion of the isolated DNA (to convert non-methylated cytosines to uracils, leaving methylated cytosines intact).
In this report, we describe the technical aspects of serum sample collection, isolation of cell-free DNA from serum, bisulfite conversion, and performance of droplet digital PCR (henceforth, digital PCR) for differentially methylated INS DNA.
Methylation of cytosines by DNA methyltransferases allows for the epigenetic control of transcription at many genes. The INS gene in humans is almost exclusively expressed in islet β cells, and there appears to be a correlation between the frequency of methylation of cytosines in the INS gene to silencing of its transcription11. As such, most cell types show substantially higher frequencies of methylation of the INS gene at various cytosines compared to β cells11…
The authors have nothing to disclose.
This work was supported by National Institutes of Health grant UC4 DK104166 (to RGM). We wish to acknowledge the assistance of the Indiana Diabetes Research Center Translation Core supported by National Institutes of Health grant P30 DK097512.
Red Top Vacutainer | Beckon Dickinson | 366441 | no additive, uncoated interior, 10 ml |
Cryovial Tube | Simport | T310-3A | polypropylene, screw cap tube, any size |
QIAamp DNA Blood Mini Kit | Qiagen | 51106 | |
Poly(A) | Sigma | P9403 | disloved in TE buffer (10 mM Tris-Cl pH 8.0 + 1 mM EDTA) to 5 µg/µl |
Absolute Ethanol (200 Proof) | Fisher Scientific | BP2818-500 | |
DPBS (with CaCl and MgCl) | Sigma | D8662 | |
0.2 mL PCR 8-strip Tubes | MidSci | AVST | |
8-strip Caps, Dome | MidSci | AVSTC-N | |
EZ DNA Methylation-Lightning Kit | Zymo | D5031 | |
ddPCR Supermix for Probes (No dUTP) | Biorad | 1863024 | |
Buffer Control for Probes | Biorad | 1863052 | |
Human Unmethylated/Methylated Primer/Probe mix | Life Technologies | AH21BH1 | |
EcoR1 | New England Biolabs | R0101L | |
twin.tec PCR Plate 96, semi-skirted | Eppendorf | 951020346 | |
Pierceable Foil Heat Seal | Biorad | 1814040 | |
PX1 PCR Plate Sealer | Biorad | 1814000 | |
QX200 AutoDG Droplet Digital PCR System | Biorad | 1864101 | |
Automated Droplet Generation Oil for Probes | Biorad | 186-4110 | |
DG32 Cartridge for Automated Droplet Generator | Biorad | 186-4108 | |
Pipet Tips for Automated Droplet Generator | Biorad | 186-4120 | |
Pipet Tip Bins for Automated Droplet Generator | Biorad | 186-4125 | |
C1000 Touch Thermal Cycler | Biorad | 1851197 | |
QX200 Droplet Reader | Biorad | 186-4003 | |
ddPCR Droplet Reader Oil | Biorad | 186-3004 |