Detection of circulating beta cell DNA in NOD mice using qMSP assay.

<p>A) Blood glucose levels in NOD mice measured every two weeks (n = 5) showing a significant rise in mean blood glucose levels after 16 weeks. The dashed line (200 mg/dL) indicates the hyperglycemic threshold. B) Pancreatic sections of the indicated groups (n = 5) were stained with H&E and the degree of insulitis was scored: no insulitis (white), peri-insulitis (dotted), mild insulitis (hatched), and invasive insulitis (black). C) In parallel, circulating beta cell DNA was measured by qMSP in each mouse group (n = 5) at designated time points. Fold changes in unmethylation are quantified by calculation of the Relative Unmethylation Ration (RUR) for each sample (see Experimental Design and Methods). The data display the mean ± standard error mean (SEM) of three independent measurements. The statistical significance was calculated by unpaired <i>t</i> tests compared with week 8 values and indicated by asterisks (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001).</p

Tissue-Specific Methylation of Human Insulin Gene and PCR Assay for Monitoring Beta Cell Death

<div><p>The onset of metabolic dysregulation in type 1 diabetes (T1D) occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP) assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD) mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.</p></div

Quantitative MSP for monitoring beta cells in islet transplant patients.

<p>(A) Blood samples were collected from 6 normal healthy individuals and pre- and post- transplantation (at days 1 and 14 post-TX) from 6 islet transplant patients. Genomic DNA from the samples was bisulfite-converted and used for nested qMSP and BSP assays. The data display the mean ± SEM of the Relative Unmethylation Ratio (RUR) calculations. (B) Plasma samples were prepared from the islet recipient blood samples and analyzed as in A. The statistical significance was calculated with the Wilcoxon test to compare RUR of samples after transplant with that before transplant and significance level indicated by asterisks (*, p<0.05; **, p<0.01).</p

Beta cell specificity of MSP.

<p>Serial dilutions of the bisulfite-converted gDNA obtained from human islets, blood, spleen and colon were used as a template for nested PCR using either BSP (A) or MSP (B) in the first-step reaction. The products were used as a template for the second-step MSP reaction. The data display the mean ± SEM of the Relative Unmethylation Ratio (RUR). The cloned <i>INS</i> promoter was used for normalization and standardization of the results. Statistically significant differences at each DNA concentration between islets and other tissues were calculated using two way ANOVA and the significance level indicated by asterisks (****, p<0.0001; ***, p<0.001; **, p<0.01).</p

The amplification efficiency of qMSP and qBSP standard curves.

<p>MSP  =  methylation-specific PCR, BSP  =  bisulfite-specific PCR, Slope  =  slope of the standard curve, R² =  the square of the correlation coefficient of the standard curve.</p

Primer selection and analytical performance of methylation-specific PCR.

<p>A) Schematic illustration of the human <i>INS</i> gene promoter region showing the position of the nine CpG sites. Solid arrows represent the bisulfite-specific primers (BSPs) that amplify both methylated and unmethylated DNA. Dashed arrows represent methylation-specific primers (MSPs) that amplify unmethylated DNA only. B) Unmethylated plasmid was serially diluted after bisulfite conversion and analyzed by qMSP using selected primer sets. Agarose gel electrophoresis of MSP reactions showing the size of the PCR products. C) Graphs of real-time SYBR Green PCR data showing linearity of C_q versus log copy number of unmethylated plasmid (averages and standard deviation (SD)) from 5 to 10⁶ copies.</p

Tissue-specific methylation of the human <i>INS</i> promoter.

<p>Genomic DNA samples obtained from human blood, breast, colon, kidney, liver, lung, spleen, stomach, and human beta cells (‘Islet cell fraction’≈70% beta cells) were analyzed for methylation of the <i>INS</i> promoter. The positions of the nine CpG sites relative to the transcription starting site (TSS) are indicated. The bars display the position and the percentage of unmethylation (white bars) to methylation (black bars) for each CpG. Each pattern results from 20 to 61 clones and obtained from 6 individuals for blood and 3 individuals for other tissues. Statistics were done using the QUMA computer program and Fisher exact test comparing each site with the same site in beta cells. The statistical significance is indicated by asterisks (*, p<0.1; **, p<0.01).</p

Tissue methylation pattern of the human <i>INS</i> exon 2.

<p>The human tissues shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094591#pone-0094591-g002" target="_blank">Figure 2</a> were analyzed for methylation of <i>INS</i> exon 2 (8 CpG), intron 1 (2 CpG) and intron 2 (2 CpG). The positions of the twelve CpG sites relative to the transcription starting site (TSS) are indicated. The bars display the position and percentage of unmethylation (white bars) to methylation (black bars) for each CpG. Each pattern results from 5 to 35 clones obtained from 5 individuals blood, 2 individuals beta cells and breast, and 3 individuals for the other tissues. Statistics were done using the QUMA computer program and Fisher exact test comparing each site with the same site in beta cells. The statistical significance is indicated by asterisks (*, p<0.1; **, p<0.01).</p

Development of a Quantitative Methylation-Specific Polymerase Chain Reaction Method for Monitoring Beta Cell Death in Type 1 Diabetes

<div><p>DNA methylation is a mechanism by which cells control gene expression, and cell-specific genes often exhibit unique patterns of DNA methylation. We previously reported that the mouse insulin-2 gene (<em>Ins2</em>) promoter has three potential methylation (CpG) sites, all of which are unmethylated in insulin-producing cells but methylated in other tissues. In this study we examined <em>Ins2</em> exon 2 and found a similar tissue-specific methylation pattern. These methylation patterns can differentiate between DNA from insulin-producing beta cells and other tissues. We hypothesized that damaged beta cells release their DNA into circulation at the onset of type 1 diabetes mellitus (T1DM) and sought to develop a quantitative methylation-specific polymerase chain reaction (qMSP) assay for circulating beta cell DNA to monitor the loss of beta cells. Methylation-specific primers were designed to interrogate two or more CpG in the same assay. The cloned mouse <em>Ins2</em> gene was methylated <em>in vitro</em> and used for development of the qMSP assay. We found the qMSP method to be sensitive and specific to differentiate between insulin-producing cells and other tissues with a detection limit of 10 copies in the presence of non-specific genomic DNA background. We also compared different methods for data analysis and found that the Relative Expression Ratio method is the most robust method since it incorporates both a reference value to normalize day-to-day variability as well as PCR reaction efficiencies to normalize between the methylation-specific and bisulfite-specific components of the calculations. The assay was applied in the streptozotocin-treated diabetic mouse model and detected a significant increase in circulating beta cell DNA before the rise in blood glucose level. These results demonstrate that this qMSP assay can be used for monitoring circulating DNA from insulin-producing cells, which will provide the basis for development of assays to detect beta cell destruction in early T1DM.</p> </div

Oligonucleotides used in this study.

<p>Underline indicates extra nucleotides added at 5′-end of the primer.</p