Abstract
In type 1 diabetes (T1D), β-cell loss is silent during disease progression. Methylation-sensitive quantitative real-time PCR (qPCR) of β-cell-derived DNA in the blood can serve as a biomarker of β-cell death in T1D. Amylin is highly expressed by β-cells in the islet. Here we examined whether demethylated circulating free amylin DNA (cfDNA) may serve as a biomarker of β-cell death in T1D. β cells showed unique methylation patterns within the amylin coding region that were not observed with other tissues. The design and use of methylation-specific primers yielded a strong signal for demethylated amylin in purified DNA from murine islets when compared with other tissues. Similarly, methylation-specific primers detected high levels of demethylated amylin DNA in human islets and enriched human β-cells. In vivo testing of the primers revealed an increase in demethylated amylin cfDNA in sera of non-obese diabetic (NOD) mice during T1D progression and following the development of hyperglycemia. This increase in amylin cfDNA did not mirror the increase in insulin cfDNA, suggesting that amylin cfDNA may detect β-cell loss in serum samples where insulin cfDNA is undetected. Finally, purified cfDNA from recent onset T1D patients yielded a high signal for demethylated amylin cfDNA when compared with matched healthy controls. These findings support the use of demethylated amylin cfDNA for detection of β-cell-derived DNA. When utilized in conjunction with insulin, this latest assay provides a comprehensive multi-gene approach for the detection of β-cell loss.
Highlights
In type 1 diabetes (T1D), β-cell loss is a silent process which leads to the development of hyperglycemia
Since demethylation index (DMI) values are determined in cell-free serum, we examined the DMI levels of amylin in whole blood preparations from healthy mice
These results identify a β-cell population which remains amylin positive while losing insulin expression and demonstrate the ability of methylation-specific primers to detect an increase in amylin circulating free amylin DNA (cfDNA) at the time of disease presentation in the non-obese diabetic (NOD) model of T1D
Summary
In type 1 diabetes (T1D), β-cell loss is a silent process which leads to the development of hyperglycemia. Our laboratory and others have previously showed the utility of differentially methylated insulin DNA as a biomarker of β-cell loss in patients and animals with T1D [8,9,10,11,12]. Examination of β-cell derived insulin cfDNA levels revealed an increase in total β-cell DNA in serum of the nonobese diabetic (NOD) mouse model of T1D and in patients with recent onset type 1 diabetes [8, 10]. The specific expression of amylin in β-cells suggests that gene expression may be regulated by methylation, making it a viable candidate for use with our assay in the detection of β-cell death. We show differential methylation of the amylin gene in insulinoma cells and primary islets of murine origin, suggesting that amylin demethylation can be used as a biomarker of β-cell loss in circulation. In T1D patients, amylin cfDNA is increased following disease onset demonstrating the utility of this biomarker in human disease
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