A methylation-sensitive multiplex droplet digital PCR assay to quantify beta-cell destruction in type 1 diabetes

This abstract has open access
Abstract Summary
Background
Detection of beta-cell methylation specific cell-free DNA (cfDNA) offers a potential biomarker to monitor beta-cell death. Measuring multiple beta-cell specific targets should increase assay sensitivity and specificity. The aim of this study was to develop a robust, high throughput, multiplex assay to track beta-cell destruction in type 1 diabetes (T1D).
Methods
Islet and PBMC DNA methylation patterns were examined using the genome-wide Illumina EPIC methylation-Bead Chip. In addition to targeting INS, two differentially methylated CpG sites were selected for triplex ddPCR assay development. Amplification specificity and sensitivity was examined in methylation specific control DNAs as well as DNA from the beta cell line EndoβH1, human islets and selected human tissues. In addition, the stability of methylation specific targets in the beta cell line EndoβH1 was examined under hyperglycaemic and proinflammatory conditions.
Serum was available from recent-onset T1D children (n=11, age-range: 4-14 years, mean age: 8.5 years, duration-range: 0.16-12.39 months,) and age-matched healthy controls (n=12, age-range: 6-15, mean-age: 9.75 years). Plasma collected in dedicated cfDNA tubes, was available from “at-risk” iarelatives of T1D patients (n=7, age range 9-50 years; mean age: 23.7 years), age and gender-matched controls (n=4; age range 8-48 years, mean age 21.5 years). cfDNA was isolated and bisulfite converted prior to absolute quantification using droplet digital PCR (ddPCR). Data were analysed by Mann-Whitney U-testing; p less than 0.05 was considered significant.
Results
Comparison of differentially methylated regions (DMRs) between islets and PBMCs identified 8856 CpG sites reaching epigenome-wide significance (P<10E-10). Comparison of islet, pancreas, liver, lung, thymus, spleen and aorta methylomes identified 653 beta-cell specific DMR CpG sites (hypomethylated=425; hypermethylated=228). Targets selected for inclusion in the multiplex assay were unaffected by hyperglycaemia or pro-inflammatory conditions. The multiplex assay discriminated recent-onset cases and at-risk relatives from controls (p less than 0.01 for all), increasing sensitivity 4 fold compared with analysing INS alone.
Submission ID :
IDS59192
Submission Type
Poster
Abstract Topics
Poster Session C
Miss Fatma Al Rashidi
University of Bristol
Dr Jody Ye
Albert Einstein College of Medicine
Dr Danijela Tatovic
Cardiff University
Professor Colin Dayan
Cardiff University
Mr Alistair Williams
University of Bristol
Dr Kathleen Gillespie
University of Bristol

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8 visits

KEY DATES

Event dates:
Thursday 25 October - Monday 29 October 2018

Abstract submission deadline:
Monday 14 May 2018

Abstract notification:
July 2018

Early registration deadline:
Monday 3 September 2018

Registration deadline:
Monday 15 October 2018

Contact
British Society for Immunology
+44 (0)20 3019 5901
congress@immunology.org