Analysis of fragment size distribution of cell-free DNA: A potential non-invasive marker to monitor graft damage in living-related liver transplantation for inborn errors of metabolism

Abstract

Graft-derived-cell-free DNA (Gcf-DNA) in plasma is a promising biomarker to monitor graft-rejection after liver transplantation (LTx). However, current methods of measuring Gcf-DNA have several limitations including high cost, long turnaround-time and the need to request donor's genetic information. In this study, eleven patients diagnosed with different inborn errors of metabolism (IEMs) who required living-related LTx were enrolled in order to establish a potentially useful noninvasive method to monitor graft damage. Circulating cell-free DNA (cfDNA) was extracted from plasma specimens serially collected at specific time points (day 0, day 1, day 7, day 14, day 30, day 60) after LTx. The distribution of Gcf-DNA fragment sizes was measured using sequencing read lengths and quantified by using Y-chromosome capture methodology in seven sex-mismatched recipients. In the analysis of fragment size distribution, we observed Gcf-DNA exhibited smaller fragment sizes than the recipient-cfDNA. Based on this phenomenon, two fragment sizes (105-145 bp, 160-170 bp) of the cfDNA pool were extracted to enrich Gcf-DNA. Accordingly, the ratio of short fragments to long fragments (S/L-Frag) in cfDNA was calculated. A high S/L-Frag ratio pointed towards an early trend of graft injury when compared to two routine liver function enzymes (ALT and AST) and Gcf-DNA, and it significantly correlated with ALT (P < 0.0001) and AST (P < 0.0001) during full-blown rejection. In conclusion, we established the Gcf-DNA size profile in patients who have undergone living-related LTx and established a potential biomarker to monitor graft function after LTx.