Measurement of Differentially Methylated INS DNA Species in Human Serum Samples as a Biomarker of Islet β Cell Death

Summary

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.

Abstract

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.

Introduction

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 therapies^3,4. 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 cells^6-9. 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.

Protocol

Ethics Statement: Protocols were approved by the Indiana University Institutional Review Board. Parents of subjects provided written informed consent, and children older than 7 years provided assent for their participation.

1. Serum Processing

NOTE: The assay as described has been rigorously tested using human serum isolated as follows.

1. Collect blood in one red top (no-additive; uncoated) blood collection tube.
2. Let sit at room temperature for 30 min to allow the clot to form.
3. Centrifuge tube at 2,000 x g for 10 min at room temperature. Transfer the supernatant (serum) to a new tube designated for serum storage (polypropylene screw top tubes, size dependent on samples).
4. Store samples at -80 ºC until ready for DNA isolation.

2. Serum DNA Extraction

NOTE: DNA is extracted with a DNA extraction kit using 50 µl of serum (recommended), following manufacturer's protocol with some modifications.

1. Prepare 200 µl lysis buffer + 1 µl poly(A) (5 µg) per sample in an microcentrifuge tube. Vortex for 15 sec. Set aside until Step 2.3.
2. Take serum samples from -80 ºC freezer and thaw at room temperature. Bring serum sample volume up to 200 µl total using phosphate-buffered saline (PBS, with calcium chloride and magnesium chloride).
3. Lyse the sample by adding 20 µl protease (1.4 Anson units/ml) to sample, followed by 200 µl lysis buffer/polyA mix (from Step 2.2) and vortex for 8 sec. Incubate sample at 56 °C for 10 min, then briefly centrifuge for 7 sec at top speed in a microcentrifuge (>10,000 x g).
4. Precipitate DNA by adding 230 µl 100% ethanol to sample and vortex for 8 sec. Briefly centrifuge for 7 sec at top speed (>10,000 x g).
5. Apply the DNA to the spin column by adding 600 µl sample mixture to mini spin column. Centrifuge at 6,000 x g for 1 min. Discard the flow-through and place column in a clean tube.
6. Wash the DNA by adding 500 µl wash buffer 1 to the column. Centrifuge at 6,000 x g for 1 min. Discard the flow-through and place column in a clean tube. Add 500 µl wash buffer 2 to column and centrifuge at top speed (>10,000 x g) for 3 min. Place column in a new 1.5 ml microcentrifuge tube and centrifuge at top speed again for 1 min.
7. Elute the DNA by placing column in a new 1.5 ml microcentrifuge tube and adding 60 µl elution buffer directly on the filter. It is important to switch tips between samples to prevent cross contamination. Incubate at room temperature for 5 min, then centrifuge at 6,000 x g for 1 min. Add another 60 μl of elution buffer and centrifuge at 6,000 x g for 1 min. 
8. Store DNA at -20 ºC for later use, or proceed immediately to bisulfite conversion.

3. Bisulfite Conversion

NOTE: Bisulfite conversion is performed using a bisulfite conversion kit, following the manufacturer's protocol with some modifications.

1. To convert unmethylated cytosines to uracils, add 130 µl of bisulfite conversion reagent to 20 µl of DNA from step 2.7 in a PCR tube (single 0.2 ml PCR tube or 8-strip PCR tubes). Save the remaining 40 µl of DNA for future use, or use in replicate reactions. Mix 20 times by pipetting up and down and centrifuge briefly to make sure no drops are on sides or lid. Incubate in a water bath or thermal cycler as follows: 98 °C for 8 min, 54 °C for 60 min, 4 °C for 5 min.
   1. Proceed to step 3.2 or store at 4 °C for up to 20 hr.
2. Add 600 µl of binding buffer to spin column and place the column in the provided collection tube. Apply the bisulfited DNA to column and mix by pipetting up and down 10 times. Centrifuge at top speed (>10,000 x g) for 30 sec. Discard flow-through.
3. Wash DNA by adding 100 µl of wash buffer to column and centrifuge at top speed (>10,000 x g) for 30 sec.
4. Perform desulphonation to remove sulphonate group to finalize the conversion of unmethylated cytosines to uracil by adding 200 µl of desulphonation buffer to the column. Let stand at room temperature for 20 min, then centrifuge at top speed (>10,000 x g) for 30 sec.
5. Wash DNA by adding 200 µl of wash buffer to the column and centrifuge at full speed for 30 sec. Discard the flow-through and repeat this wash step.
6. Place the column into a 1.5 ml microcentrifuge tube. Elute DNA by adding 10 µl elution buffer directly to column filter. Incubate for 1 min. Centrifuge for 30 s at full speed to elute DNA. Quantify recovered DNA by spectrophotometry at A260.
   NOTE: The DNA detected by spectrophotometry represents primarily the carrier polyA DNA that was added at step 2.3 above. Typically, recovery of polyA is ≥85% of the input (>4.25 µg).
7. Store DNA at -20 °C until ready to proceed with digital PCR.

4. Multiplex Digital PCR

1. Make a master mix with enough solution for each sample and control. Include at least one sample containing water (negative control), one sample of a plasmid containing unmethylated INS (1 pg, positive control), one sample of plasmid containing methylated INS (1 pg, positive control), and one sample containing a 1:1 mixture of plasmids.
   1. To prepare the PCR master mix, add 12.5 µl per reaction of PCR buffer (e.g., 125 µl per 10 samples), 1.25 µl per reaction primer/probe mix (e.g., 12.5 µl per 10 samples), 8.25 µl per reaction water (e.g., 82.5 µl per 10 samples), 0.5 µl per reaction EcoR1 enzyme (e.g., 5 µl per 10 samples).
      NOTE: Primers and probes used here are as follows: Forward Primer: 5'-GGAAATTGTAGTTTTAGTTTTTAGTTATTTGT-3'; Reverse Primer: 5'-AAAACCCATCTCCCCTACCTATCA-3'; FAM probe: FAM-5'-ACCCCTACCACCTAAC-3'-MGB; VIC probe: VIC-5'-ACCCCTACCGCCTAAC-3'-MGB.
2. Set up multiplex PCR reaction in a 96-well plate. Add 19.5 µl of master mix to each well. Add 2.5 µl of bisulfite converted DNA sample into each appropriate well (save the remaining 7.5 µl of bisulfite converted DNA or use in replicate reactions). Mix by pipetting up and down several times.
   NOTE: All 8 wells in a row must contain sample or buffer control.
3. Seal with a foil seal using a plate sealer and centrifuge in a plate spinner until there is no liquid on the sides of the wells.
4. Set up the automated droplet generator on the touchscreen.
   NOTE: All 8 wells in the cartridge must contain sample or buffer control.
   1. Set the number of rows being used by touching the row(s) in which samples are loaded into on the 96-well plate from step 4.2 and highlight the row in blue to indicate an active row.
   2. Load consumables from back to front to avoid contamination. To start, add cartridges along the back row of the instrument. Load tips into center row of the instrument.
      NOTE: The cartridges can only fit into the holders in the correct orientation.
   3. Place 96-well plate into the instrument. Remove a cold block from -20 °C freezer and place a new skirted 96-well plate into it. Place it inside the instrument next to the sample loaded 96-well plate.
   4. Add oil to dispenser in the front of the instrument. Select type of oil on touchscreen.
   5. Touch the blue start button to start the run. Confirm plate setup and touch start run button to begin.
5. Once finished, remove the 96-well plate that contains the newly formed droplets and seal with a foil seal using a plate sealer.
6. Perform PCR₁₀ on a thermal cycler using the following program: 95 °C for 10:00 min; 40 cycles at: 94 °C for 00:30 min, 57.5 °C for 01:00 min; 98 °C for 10:00 min; 12 °C for 10 min or up to 24 hr.
7. Place the plate on the droplet reader and set up the reader.
   1. Click on the setup tab. Open a new template by clicking template > new. Enter a file name.
   2. Use the well editor to set the parameters for each sample. Give each sample a unique name, set experiment to Rare Event Detection (R-RED), and set master mix to the mix used in step 4.1.
   3. Give Target 1 a name (i.e., unmethylated) and set Target 1 as unknown (U). Give Target 2 a name (i.e., methylated) and set Target 2 as reference (R).
   4. Click Run tab to start the run. In the run options window, select the detection chemistry (FAM/VIC).

5. Data Analysis

1. Open results in analyze tab and analyze using 2-D plot based on positive controls (see Figure 1).
2. Export CSV file generated by droplet reader to a spreadsheet program for data analysis.
3. To generate "copies per µl" of serum, use the formula: (concentration*250)/(volume of serum used for DNA isolation in µl).⁶
   NOTE: The data is typically converted to log₁₀ when acquired from human serum to ensure normal distribution. Alternatively, data can be plotted on a log₁₀ scale and analyzed by non-parametric statistics.

Results

To interpret data appropriately, we use plasmid controls for both the unmethylated and methylated target INS DNA in each digital PCR run. These controls ensure that signals corresponding to methylated and unmethylated DNA are clearly distinguishable. Figure 1 shows the 2-D scatter plots corresponding to droplets for plasmid controls containing bisulfite-converted unmethylated INS DNA (Figure 1A), methylated INS DNA (Figu...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 CaCl2 and MgCl2)	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