BRIEF REPORT: Elevations in Circulating Methylated and Unmethylated Preproinsulin DNA in New-Onset Type 1 Diabetes Abbreviated title: Elevated circulating preproinsulin DNA in T1D

Elevated ratios of circulating unmethylated to methylated preproinsulin (INS) DNA have been suggested to reflect β-cell death in type 1 diabetes (T1D). We tested the hypothesis that absolute levels (rather than ratios) of unmethylated and methylated INS DNA differ between new-onset T1D subjects and controls, and assessed longitudinal changes in these parameters. We used droplet digital PCR to measure levels of unmethylated and methylated INS DNA in serum from subjects at T1D onset, and at 8 weeks and one year post-onset. Compared to controls, levels of both unmethylated and methylated INS DNA were elevated at T1D onset. At 8 weeks post-onset, methylated INS DNA remained elevated, but unmethylated INS DNA fell. At one year post-onset, both unmethylated and methylated INS DNA returned to control levels. Subjects with obesity, type 2 diabetes (T2D), and autoimmune hepatitis exhibited lower levels of unmethylated and methylated INS compared to T1D subjects at-onset and no differences compared to controls. Our study shows that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. Our work emphasizes the need to consider absolute levels of differentially methylated DNA species as potential biomarkers of disease.