Detection of islet β-cell death in vivo by multiplex PCR analysis of differentially methylated DNA

Endocrinology. 2013 Sep;154(9):3476-81. doi: 10.1210/en.2013-1223. Epub 2013 Jul 3.


Noninvasive detection of early β-cell death in type 1 diabetes might identify individuals in whom therapeutic interventions would preserve β-cell mass and prevent hyperglycemia. Recent studies in mice have shown that β-cell death produces a corresponding increase in unmethylated preproinsulin (PPI) DNA in serum. Here, we report the development of a novel assay using dual fluorescent-probe multiplex PCR (TaqMan) to detect differential methylation of circulating PPI DNA. Key assay features include low background signals, linear assay output across a large range of values, and simultaneous detection of methylated and unmethylated PPI DNA in a single reaction. We defined the "unmethylation index" as a summary parameter that reflects the relative amounts of unmethylated vs methylated PPI DNA. To validate this assay's ability to detect β-cell death in vivo, we measured the unmethylation index in the serum of diabetic mouse models, including high- and multiple low-dose streptozotocin-induced diabetes, and the nonobese diabetic mouse model of type 1 diabetes. Our data show a significantly increased unmethylation index concordant with the known timeline of β-cell death that precedes the onset of hyperglycemia. Subsequently, we observed a decrease in the unmethylation index following diabetes development, likely reflecting the absence of further β-cell death in the pancreas. We conclude that simultaneous measurement of methylated and unmethylated PPI DNA using the multiplex PCR method described here is a readily available and sensitive indicator of dying β-cells that may be useful to track diabetes progression and response to therapeutic intervention.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Validation Study

MeSH terms

  • Algorithms
  • Animals
  • Apoptosis
  • Cell Death*
  • DNA / blood*
  • DNA Methylation*
  • Diabetes Mellitus, Type 1 / blood
  • Diabetes Mellitus, Type 1 / diagnosis*
  • Diabetes Mellitus, Type 1 / pathology
  • Disease Models, Animal*
  • Female
  • Insulin / blood*
  • Insulin / genetics
  • Insulin-Secreting Cells / pathology*
  • Male
  • Mice
  • Mice, Inbred Strains
  • Mice, SCID
  • Multiplex Polymerase Chain Reaction
  • Necrosis
  • Protein Precursors / blood*
  • Protein Precursors / genetics
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Specific Pathogen-Free Organisms


  • Insulin
  • Protein Precursors
  • preproinsulin
  • DNA