Detection of β cell death in diabetes using differentially methylated circulating DNA

Abstract

In diabetes mellitus, β cell destruction is largely silent and can be detected only after significant loss of insulin secretion capacity. We have developed a method for detecting β cell death in vivo by amplifying and measuring the proportion of insulin 1 DNA from β cells in the serum. By using primers that are specific for DNA methylation patterns in β cells, we have detected circulating copies of β cell-derived demethylated DNA in serum of mice by quantitative PCR. Accordingly, we have identified a relative increase of β cell-derived DNA after induction of diabetes with streptozotocin and during development of diabetes in nonobese diabetic mice. We have extended the use of this assay to measure β cell-derived insulin DNA in human tissues and serum. We found increased levels of demethylated insulin DNA in subjects with new-onset type 1 diabetes compared with age-matched control subjects. Our method provides a noninvasive approach for detecting β cell death in vivo that may be used to track the progression of diabetes and guide its treatment.

Fig. 1.

DNA from βTC3 and PMJ cell lines and β and non-β cells have a differentially methylated CpG dinucleotide in the Ins1 gene. (A) (Upper) Unmodified DNA sequence of murine Ins1 DNA depicting the position of the differentially methylated CpG dinucleotide (arrow). (Lower) Comparison of bisulfite treated genomic DNA from either the βTC3 or PMJ cell line, demonstrating the nucleotide modification of CpG dinucleotides owing to demethylation at position 523393278. (B) Sequence analysis of product amplified in the first-step PCR. The sequence of 15 clones from murine β cells and 8 clones from murine liver cells are shown (○ indicates demethylated cytosines; ●, methylated cytosines). The locations of the methylation sites from the transcription start site are indicated. The primers of the second-step PCR were specific for methylated/demethylated cytosine at bp +177, corresponding to nucleotide 52339278.

Fig. 2.

Identification of differentially methylated DNA by real-time PCR. Bisulfite-treated purified DNA from tissues, cells, or serum origin was purified and used in the first-step, methylation-insensitive reaction. The products were gel-purified and used as a template in a second-step reaction with methylation-specific primers.

Fig. 3.

Demethylated Ins1 DNA is enriched in primary islets and FACS-sorted primary insulin-positive cells. (A) Ratio of demethylated:methylated DNA in primary murine tissues. The cycle differences were normalized to the cycle difference of kidney DNA. The data are from a single experiment representative of more than five experiments. (B) FACS plot analysis showing the presence of insulin-positive and -negative cells sorted from dispersed islets. (C) Demethylated:methylated DNA levels in the sorted cell population (shown in B).The insulin-positive cell cycle difference was normalized to the insulin-negative cell cycle difference. Data are from a single experiment representative of two experiments. (D) DNA from the first-step reactions from sorted β cells and from islet-derived non-β cells were mixed in ratios of 1:1, 1:10, and 1:100 and then added to the second-step reaction. The relationship between the ratio of DNA and the demethylation index is shown (r² = 0.96; P = 0.0038).

Fig. 4.

Demethylated Ins1 DNA is increased in the serum after STZ treatment of mice. (A) Blood glucose levels of untreated and STZ-injected BALB/c mice (n = 6 animals per group) *P < 0.05; ^±P < 0.02 vs. prediabetic mice. (B) Demethylation index of the nested PCR performed on DNA from sera of the BALB/c mice. Between 16 and 18 mice were analyzed at each time point. The sera from two mice were pooled for analysis. *P < 0.05. The box-and-whisker plots show the minimum and maximum values. (C) Histomorphic analysis of DAPI-positive, insulin-positive cells in the islets of the STZ-treated mice shown in ^B. *P < 0.0001; ^±P < 0.002. (D) Representative islets of STZ-treated mice. DAPI is in blue; insulin, in red.

Fig. 5.

Serum-derived demethylated Ins1 DNA is increased in prediabetic NOD mice with impaired glucose tolerance. (A) IPGTT data for prediabetic NOD mice at various ages (n ≥5 per group). Note that the fasting glucose (at t = 0) is similar at all time points. (B) Area under the curve of IPGTT data from A. *P < 0.05. (C) Demethylation index measured with DNA from the sera of prediabetic (wk 7–14) and diabetic NOD mice. P = 0.0002 by ANOVA; **P < 0.01; *P < 0.05; n = 5, 5, 5, 7, and 5 mice/group. The box-and-whisker plots show the minimum and maximum values. (D and E) In a separate experiment, pancreata and serum were harvested from mice at the indicated ages (n = 5 mice per time point) for measurement of insulin content and demethylation index, respectively. The insulin content and demethylation index in 11- and 15-wk-old mice were compared with 7-wk-old mice. *P < 0.05; **P < 0.02 by post hoc analysis of ANOVA. (E) Relationship between pancreatic insulin content and demethylation index in individual mice. Two measurements from each mouse are plotted (r² = 0.28; P < 0.05).

Fig. 6.

Analysis of insulin DNA sequences in human tissues and sera. (A) Unmodified DNA sequence in human Ins gene showing preserved CpG pair at bp +273 and +399 identified in the UCSC Genome Browser (http://genome.ucsc.edu/cgi-bin/hgGateway). (B) Sequence data of the first-step PCR showing methylation DNA patterns in primary human kidney and whole islets. The arrow shows the presence of demethylated CpG found in human islets at bp +399 (at position 2182036, site of the reverse primer). Note the two peaks in human islets representing both demethylated and methylated forms from β cells and non-β cells in the islets. (C) Sequence analysis of product amplified in the first-step PCR from sorted human β cells and kidney. The sequence of 10 clones from human β cells and 12 clones from human kidney cells are shown (○ indicates demethylated cytosines; ●, methylated cytosines). The base pairs are indicated downstream from the transcription start site. The primers of the second-step PCR were specific for methylated/demethylated cytosine at bp +273 and +399. (D) DNA was isolated from human kidney, liver, and islets and analyzed by nested PCR. Synthetic DNA was also analyzed in these reactions. Each dot represents a separate isolation and analysis of tissue DNA. The demethylation index was significantly greater with DNA from islets compared with liver and kidney. ***P < 0.001. (E) DNA was isolated from five subjects with recent-onset T1D (●) and from six healthy control subjects (■). The demethylation index was significantly higher in patients with T1D (P = 0.017, Mann–Whitney U test).