Genome-Wide Cell-Free DNA Methylation Analysis Reveals Significant Recipient Tissue Injury in Allograft Rejection

Abstract

<h3>Purpose</h3> Acute rejection (AR) causes significant allograft injury. However, its systemic effect on recipient tissues remains poorly defined. Donor-derived cell-free DNA (ddcfDNA) is a sensitive marker of allograft rejection, but does not capture any concurrent recipient tissue injury. Herein, we leverage cfDNA epigenetics as marker of tissue injury to profile recipient tissue injury associated with AR. <h3>Methods</h3> cfDNA was extracted in 92 plasma samples from 33 heart transplant recipients (HTR) (19 with AR and 14 stable controls) from the GRAfT study (NCT 02423070), and 19 healthy controls (HC). <b>Measurements:</b> Total cell-free nuclear (ncfDNA) and mitochondrial DNA (mtcfDNA) were assayed via digital-droplet PCR. To quantify recipient cfDNA, we first measured ddcfDNA fraction by shotgun sequencing; recipient cfDNA is the difference of total ncfDNA and ddcfDNA. Next, we performed whole-genome bisulfite sequencing on cfDNA and used tissue-specific DNA methylation signatures to deconvolute recipient tissue sources of cfDNA. Median [interquartile range (IQR)] of cfDNA percent or copies (cp)/mL are reported. <h3>Results</h3> ddcfDNA levels increased 6 fold with AR compared to stable HTR (0.54 [0.32-1.21] % vs. 0.09 [0.03 - 0.12] %, <i>p</i><0.01). ncfDNA, which is >99% recipient-derived, was 5X higher in stable HTR (7997 [3974-9740] cp/mL) than HC (1224 [921-1985] cp/mL, <i>p</i><0.01). With AR, ncfDNA doubled (14949 [8460-24018] cp/mL) compared with stable HTR (<i>p</i><0.01). mtcfDNA tripled with AR than stable HTR, and showed excellent discriminatory performance (AUC = 0.92, 95%CI [0.84-0.99]). Genome-wide methylation analysis showed significantly higher cfDNA from hematopoietic cells, vascular endothelium, hepatocyte and kidney with AR than Stable HTR, Fig.1. <h3>Conclusion</h3> Genome-wide cfDNA methylome analysis shows increased recipient tissue injury with AR. Ongoing studies intend to validate this finding and test if this approach can distinguish antibody-mediated from acute cellular rejection phenotypes.