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Multiplexing DNA methylation markers to detect circulating cell-free DNA derived from human pancreatic β cells
Daniel Neiman, … , Ruth Shemer, Yuval Dor
Daniel Neiman, … , Ruth Shemer, Yuval Dor
Published June 23, 2020
Citation Information: JCI Insight. 2020;5(14):e136579. https://doi.org/10.1172/jci.insight.136579.
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Research Article Endocrinology Metabolism

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

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Abstract

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 6 DNA methylation markers, that identifies β cell DNA in mixtures containing as little as 0.03% β cell DNA (less than 1 β cell genome equivalent). Based on this assay, plasma from nondiabetic individuals (N = 218, aged 4–78 years) contained on average only 1 β cell genome equivalent/mL. As expected, cell-free DNA (cfDNA) from β cells was significantly elevated in islet transplant recipients shortly after transplantation. We also detected β cell cfDNA in a patient with KATP congenital hyperinsulinism, in which 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 long-standing disease (>4 months, N = 38). We discuss the utility of sensitive β cell cfDNA analysis and potential explanations for the lack of a β cell cfDNA signal in type 1 diabetes.

Authors

Daniel Neiman, David Gillis, Sheina Piyanzin, Daniel Cohen, Ori Fridlich, Joshua Moss, Aviad Zick, Tal Oron, Frida Sundberg, Gun Forsander, Oskar Skog, Olle Korsgren, Floris Levy-Khademi, Dan Arbel, Saar Hashavya, A.M. James Shapiro, Cate Speake, Carla Greenbaum, Jennifer Hosford, Amanda Posgai, Mark A. Atkinson, Benjamin Glaser, Desmond A. Schatz, Ruth Shemer, Yuval Dor

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Figure 1

Procedure for analysis of β cell–specific methylation markers.

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Procedure for analysis of β cell–specific methylation markers.
(A) Proce...
(A) Procedure for multiplex amplification of methylation markers. Following bisulfite treatment, loci of interest were amplified using specific primers containing 5′ 25-mer adapters. In a second PCR step, the adapters were extended to include the full adapter sequence required for Illumina sequencing, as well as a 6-bp index barcode allowing us to run multiple products simultaneously. Up to 30 distinct loci can be amplified using this 2-step PCR approach. (B) Schematic of workflow. First, cfDNA was extracted from rapidly spun plasma and treated with bisulfite to convert unmethylated cytosines to uracils. Methylation-independent PCR was used to amplify marker loci. Markers that are methylated or unmethylated only in a tissue of interest were selected based on comparative methylome analysis using an extensive matrix of tissue- and cell type–specific methylomes. The products of amplification were sequenced thousands of times on the MiSeq/NextSeq platform to determine the fraction of molecules in plasma that carry the tissue-specific methylation pattern. When the fraction was multiplied by the total concentration of cfDNA in a sample, the concentration of cfDNA molecules derived from a specific tissue was obtained.

Copyright © 2022 American Society for Clinical Investigation
ISSN 2379-3708

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