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It has been proposed that unmethylated insulin promoter fragments in plasma derive exclusively from β-cells, reflect their recent demise and can be used to assess β-cell damage in type 1 diabetes. Herein we describe an ultrasensitive assay for detection of a β-cell-specific DNA methylation signature, by simultaneous assessment of six DNA methylation markers, that identifies β-cell DNA in mixtures containing as little as 0.03% β-cell DNA (less than one β-cell genome equivalent). With this assay, plasma from non-diabetic individuals (N=218, aged 4-78 years) contained on average only one β-cell genome equivalent/ml. As expected, β-cell cfDNA was significantly elevated in islet transplant recipients shortly after transplantation. We also detected β-cell cfDNA in a patient with KATP congenital hyperinsulinism where substantial β-cell turnover is thought to occur. Strikingly, in contrast to previous reports, we observed no elevation of β-cell-derived cfDNA in autoantibody positive subjects at-risk for type 1 diabetes (N=32), individuals with recent-onset type 1 diabetes (<4 months, N=92), or those with a long-standing disease (>4 months, N=38). We discuss the utility of sensitive beta-cell cfDNA analysis and potential explanations for the lack of a β-cell cfDNA signal in T1D.

Multiplexing DNA methylation markers to detect circulating cell-free DNA derived from human pancreatic β cells