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

Martin Bizet,Emily K Sims,Silva Arslanian,François Fuks ,Jamie L Felton,Jean-Valéry Turatsinze ,Kieren J Mather,Matthieu Defrance ,Appakalai N Balamurugan,Farooq Syed,Carmella Evans‐Molina ,Marco Bugliani,Raghavendra G Mirmira,Jennifer B Nelson,Miriam Cnop,Piero Marchetti,Sarah A Tersey,Bobbie‐Jo Webb‐Robertson ,Ezio Bonifacio,Décio L Eizirik ,Anette‐Gabriele Ziegler ,Nicole Jiyun Kang

doi:10.1186/s13148-020-00906-5

Abstract

BackgroundIdentification 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.ResultsTo 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.ConclusionOur data suggest that concurrent measurement of circulating unmethylated INS and CHTOP has the potential to detect islet death in youth at risk for both T1D and T2D. Our data also support the use of multiple parameters to increase the confidence of detecting islet damage in individuals at risk for developing diabetes.

Highlights

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
Four key findings emanate from our study: (a) neither differentially methylated chromatin target of PRMT1 (CHTOP) nor Gene encoding preproinsulin (INS) exhibits specificity for the β cell within the islet, as α cells exhibit nearly the same degree of unmethylation at these genes, (b) the combination of unmethylated CHTOP-817 and INS DNA levels provides greater confidence that signals are originating from islet cells, (c) autoantibody-negative first-degree relatives (FDRs) of individuals with T1D exhibit a circulating differentially methylated DNA signature similar to individuals with T1D, and (d) youth with overweight/obesity, as a group, exhibit elevated markers consistent with islet cell death and inflammation
Our studies in humans utilized cross-sectional cohorts, and we cannot comment on the occurrence or the timing of islet cell death following initial islet assault during the progression from obesity to T2D or from antibody-negative FDR to T1D

Summary

Introduction

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. There is an alarming increase in the incidence of type 1 diabetes (T1D) and type 2 diabetes (T2D), and it is estimated that 425 million individuals are afflicted with diabetes worldwide [1] Both major forms of diabetes have been viewed as distinct: T1D develops as a result of selective destruction of pancreatic β cells by the immune system, while T2D develops secondary to insufficient insulin secretion in the context of insulin resistance in peripheral tissues. In both forms, clinical manifestations of disease occur only after substantial loss of functional β cell mass, and application of therapeutic interventions at this time point has at best had very limited success in restoring β cell mass and function. The appearance of circulating unmethylated INS does not exclusively report on β cell death, and more rigorous or complementary biomarkers are needed

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