PMC

Unmethylated INS DNA in patients with T1D. A: The levels of unmethylated INS DNA were measured in 43 subjects with recent-onset T1D and 13 nondiabetic control (Ctl) subjects of similar age (unpaired t test, ***P = 0.001). In the patients and control subjects, unmethylated C_t values ranged from 29.8 to 37.2 and from 22.3 to 37.3, respectively, and the methylated C_t values ranged from 15.4 to 30.7 and from 15.1 to 24.2. B: The C-peptide responses to a mixed meal (area under the curve [AUC]) and the corresponding Δ are shown for the subjects with recent-onset T1D (r = 0.34, P = 0.03).

Effects of teplizumab therapy on clinical responses and levels of unmethylated INS DNA. A and B: Percent decline in C-peptide (*P = 0.03) and increase in insulin use (**P < 0.009) from baseline are shown for drug- and placebo-treated subjects. The baseline C-peptide and insulin in the drug group was 0.53 ± 0.07 pmol/mL/min and 0.38 ± 0.03 units/kg/day, respectively, and in the placebo group 0.53 ± 0.08 pmol/mL/min and 0.38 ± 0.04 units/kg/day (P = 0.18 and 0.94). C: Relative β-cell–derived DNA levels before and after treatment with teplizumab or placebo. There was a significant reduction in the Δ in subjects treated with teplizumab (***P < 0.0001) but not placebo (P = 0.08). At baseline, the Δs in the drug- and placebo-treated groups were similar (P = 0.8). D: Subjects treated with teplizumab had a greater decline in the Δ (*P = 0.04).

Measurement of unmethylated INS DNA by real-time PCR. DNA extraction of sera, tissue, and cells and bisulfite treatment. A: DNA was isolated from serum and treated with bisulfite. The treated DNA was amplified using a first-step, methylation-insensitive reaction between bp 329 and 399 from the transcription start site. The products of this reaction were used in a second-step RT-PCR reaction with primers specific for methylated or unmethylated DNA sequences. The difference in the C_t values (methylated − unmethylated) was determined and is represented by the Δ. B: The methylated and unmethylated sequences from 10 clones from human β-cells, 14 clones from human islet cells, and 12 clones from human kidney cells are indicated at eight CpG sites (○, unmethylated cytosines; ●, methylated cytosines). The methylation pattern of the CpG sites in β-cells and kidney has previously been published (). The bp are indicated downstream from the transcription start site. The majority of the CpG sites in purified β-cells are unmethylated, whereas there is a mixture of methylated and unmethylated sites in the products of the first-step PCR reaction from islet DNA reflecting the mixture of endocrine cells. The products of the PCR reaction with kidney DNA are predominantly methylated. C, cytosine; G, guanine; T, thymine. CG (cytosine-guanine) refers to methylated CpG sites, and TG (thymine-guanine) refers to unmethylated CpG sites.