Characteristics of Urine Cell-Free DNA in Heart and Lung Transplant

Abstract

Purpose Plasma donor-derived cell-free DNA (ddcfDNA) is a biomarker of organ transplant rejection, but the use of urine ddcfDNA as a clinical biomarker has not been thoroughly investigated. We hypothesize ddcfDNA is present in urine with levels that are predictive of plasma ddcfDNA. To test this, we assessed the physical characteristics of urine ddcfDNA and determine its quantitative relationship to plasma ddcfDNA in heart (HT) and lung transplant(LT) recipients. Methods We evaluated %ddcfDNA in concurrently collected urine and plasma samples from 40 patients (16HT and 24LT). Pre-transplant donor and recipient blood was collected for DNA extraction and genotyping. After transplantation, we collected serial urine and plasma samples concurrently for cfDNA extraction, library construction, and shotgun sequencing. Sequence reads were separated as recipient-derived or donor-derived using single nucleotide polymorphisms from the pre-transplant genotype data. The %ddcfDNA was computed as percentage of donor reads to donor plus recipient reads. We compared fragment length, nucleotide composition, and amount of %ddcfDNA in urine and plasma cfDNA. Results 51 urine cfDNA samples were sequenced (mean depth of 30 million reads). Urine and plasma cfDNA were both Adenine/Thymine rich compared to genomic DNA (60% vs. 40%), however urine cfDNA was shorter than plasma cfDNA (peak length 80 vs. 167 base pairs). Immediately after transplant surgery, urine ddcfDNA levels were highest (x= 1.013%) and then decayed logarithmically to a baseline level of 0.176%. Plasma ddcfDNA also showed a post-transplant logarithmic decay pattern. Concurrently measured urine and plasma %ddcfDNA values showed a predictable relationship with saturation kinetics (r2=0.449) (Figure). Conclusion Urine %ddcfDNA levels were predictive of plasma %ddcfDNA levels suggesting that like plasma %ddcfDNA, urine %ddcfDNA is a potential biomarker of allograft rejection. Future studies should validate these findings and assess the clinical utility of urine %ddcfDNA.