Circulating unmethylated CHTOP and INS DNA fragments provide evidence of possible islet cell death in youth with obesity and diabetes

Abstract

Background: Identification of islet β cell death prior to the onset of type 1 diabetes (T1D) or type 2 diabetes (T2D) might allow for interventions to protect β cells and reduce diabetes risk. Circulating unmethylated DNA fragments arising from the human INS gene have been proposed as biomarkers of β cell death, but this gene alone may not be sufficiently specific to report β cell death. Results: To identify new candidate genes whose CpG sites may show greater specificity for β cells, we performed unbiased DNA methylation analysis using the Infinium HumanMethylation 450 array on 64 human islet preparations and 27 non-islet human tissues. For verification of array results, bisulfite DNA sequencing of human β cells and 11 non-β cell tissues was performed on 5 of the top 10 CpG sites that were found to be differentially methylated. We identified the CHTOP gene as a candidate whose CpGs show a greater frequency of unmethylation in human islets. A digital PCR strategy was used to determine the methylation pattern of CHTOP and INS CpG sites in primary human tissues. Although both INS and CHTOP contained unmethylated CpG sites in non-islet tissues, they occurred in a non-overlapping pattern. Based on Naïve Bayes classifier analysis, the two genes together report 100% specificity for islet damage. Digital PCR was then performed on cell-free DNA from serum from human subjects. Compared to healthy controls (N = 10), differentially methylated CHTOP and INS levels were higher in youth with new onset T1D (N = 43) and, unexpectedly, in healthy autoantibody-negative youth who have first-degree relatives with T1D (N = 23). When tested in lean (N = 32) and obese (N = 118) youth, increased levels of unmethylated INS and CHTOP were observed in obese individuals.

Cell-Free DNA Fragments as Biomarkers of Islet β-Cell Death in Obesity and Type 2 Diabetes

“…Using banked sera from subjects in the T1D Exchange registry, one study demonstrated elevations in both unmethylated INS and methylated INS in subjects from both C-peptide-positive and C-peptide-negative subjects with longstanding (≥9 years) T1D [ 37 ]. These findings seem indicative of the persistence of β cells in such subjects, where ongoing β-cell death may give rise to circulating unmethylated INS signals and ongoing systemic autoimmunity/inflammation may give rise to elevated methylated INS signals [ 38 ]. At least two studies have examined the occurrence of β-cell death in subjects at risk for T1D.…”

“…A more recent study [ 38 ] examined autoantibody-negative first-degree relatives of those with T1D (a group that is at modestly higher risk of developing T1D compared to otherwise healthy controls) and demonstrated striking elevations of unmethylated INS in this group. Although the findings of these studies suggest that a pre-symptomatic period with active β-cell death may occur, these findings were not corroborated in another study that used a DNA sequencing approach to examine unmethylated INS in subjects at-risk for T1D [ 29 ].…”

“…Apart from the INS gene, other genes have been interrogated for the ability to identify β-cell death in subjects with T1D. These include other genes that are known to be preferentially expressed in β cells ( IAPP and GCK ) [ 42 , 43 ] and another ( CHTOP ) identified from an unbiased screen [ 38 ]. In all these cases, elevations in the unmethylated DNA species were observed in subjects with T1D compared to controls, but the utility of IAPP and GCK genes, alone or in combination with other genes, in stratifying populations at-risk for T1D remains untested.…”

“…Arguably, the cohort represented by autoantibody-positivity and persistence of β-cell function are more akin to patients with early T1D, notwithstanding the presence of overweight. A more recent study by our group examined both methylated and unmethylated INS and CHTOP in obese youth across the spectrum of normal glucose tolerance to T2D; these categories included lean controls with normal glucose tolerance (NGT), overweight/obese with normal glucose tolerance (OB-NGT), overweight/obese with impaired glucose tolerance (IGT), and overweight/obese with T2D with and without evidence of autoantibodies [ 38 ]. Obese youth, as a group, showed statistically greater levels of unmethylated CHTOP and both unmethylated and methylated INS compared to lean control youth—findings suggestive of β-cell death in youth with obesity.…”

Epigenetic Regulation of Adipogenesis in Development of Metabolic Syndrome

“…The study suggested that the methylation changes in the circulating blood cells could serve as a biomarker for prediction of metabolic diseases in offsprings of women with obesity and GDM ( Hjort et al, 2018 ). A different study identified the differential methylation status of circulating cell-free CHTOP and INS1 DNA fragments as potential biomarkers for possible islet death in youths with obesity and diabetes ( Syed et al, 2020 ). A study by Nishimoto et al investigated the role of cfDNA in development adipose tissue inflammation.…”