11th International Symposium on Circulating Nucleic Acids in Plasma and Serum (CNAPS)

Comparison of two techniques for cell-free DNA detection of hepatocellular injury following liver transplantation

Daniel Cox 1,2,3 Su Kah Goh 1,2 Boris Wong 3,4 Adam Testro 5 Christopher Christophi 1,2 Vijayaragavan Muralidharan 1,2 Alexander Dobrovic 1,3,4
1Department of Surgery, University of Melbourne, Melbourne, Victoria, Australia
2HPB & Transplant Unit, Austin Health, Melbourne, Victoria, Australia
3Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Melbourne, Victoria, Australia
4School of Cancer Medicine, La Trobe University, Melbourne, Victoria, Australia
5Department of Gastroenterology & Hepatology, Liver Transplant Unit, Austin Health, Melbourne, Victoria, Australia

Graft-derived cell-free DNA (gdcfDNA) quantification is a developing tool for monitoring organ health following transplantation. Acute cellular rejection, sepsis and ischaemia following liver transplantation increase rates of graft cell death leading to elevated circulating levels of gdcfDNA. Until recently, gdcfDNA quantification assays have largely relied on donor and recipient genetic chimerism to differentiate gdcfDNA from ‘background’ cfDNA. [1, 2] Our group has previously developed a probe-free droplet digital PCR assay to quantify gdcfDNA based on the identification of small deletion/insertion polymorphisms to distinguish donor-derived cfDNA from that of the recipient. [3]

DNA methylation conforms to specific patterns depending on the tissue of origin and therefore forms another promising target to differentiate gdcfDNA from ‘background’ cfDNA. [4] Here we describe the development of a methylation-specific, droplet digital PCR, gdcfDNA quantification assay. This assay is based on the use of tissue-specific (hepatocyte) DNA methylation patterns to discriminate gdcfDNA from background cfDNA. We compare the performance of this methylation-specific assay with our previous polymorphism-dependent quantification technique and discuss the relative benefits of each approach.

  1. Beck J, Bierau S, Balzer S et al. Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury. Clinical Chemistry 2013; 59 (12): 1732-41, doi: 10.1373/clinchem.2013.210328
  2. Bruno D, Ganesamoorthy D, Thorne N et al. Use of copy number deletion polymorphisms to assess DNA chimerism. Clinical Chemistry 2014, 60(8): 1105-14, doi: 10.1373/clinchem.2013.216077
  3. Goh SK, Do H, Muralidharan V, et al. Donor-Specific Cell-Free DNA as an Emerging Biomarker of Organ Rejection after Liver Transplantation. Transplantation 2018; 102: S182, doi: 10.1097/01.tp.0000542826.68057.b6
  4. Moss J, Magenheim J, Neiman D et al. Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease. Nature Communications 2018, 9: 5068, doi: 10.1038/s41467-018-07466-6









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