Circulating Differentially Methylated Amylin DNA as a Biomarker of β-Cell Loss in Type 1 Diabetes.
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.
Research Institute, Islet Biology, Winthrop-University Hospital, Mineola, NY, United States of America.
John A
JA
Olsen
Research Institute, Islet Biology, Winthrop-University Hospital, Mineola, NY, United States of America.
Lauren A
LA
Kenna
Research Institute, Islet Biology, Winthrop-University Hospital, Mineola, NY, United States of America.
Michael G
MG
Spelios
Max McGee National Research Center for Juvenile Diabetes, Children's Research Institute of Children's Hospital of Wisconsin, Milwaukee, WI, United States of America.|Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, United States of America.
Martin J
MJ
Hessner
Research Institute, Islet Biology, Winthrop-University Hospital, Mineola, NY, United States of America.|Stony Brook University School of Medicine, Stony Brook, NY, United States of America.
Eitan M
EM
Akirav
D000293:Adolescent; D000818:Animals; D001402:B-Lymphocytes / Q000473:pathology*; D015415:Biomarkers / Q000378:metabolism*; D002648:Child; D019175:DNA Methylation; D003922:Diabetes Mellitus, Type 1 / Q000473:pathology*; D005260:Female; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000235:genetics*; D008297:Male; D051379:Mice
D015415:Biomarkers; D058228:Islet Amyloid Polypeptide