Deconvolution of Donor and Recipient Transcripts from Frozen Lung Transplant Biopsies

Abstract

Despite years of study using conventional approaches applied to human and animal models, the biology of lung transplantation remains incompletely understood. We aimed to develop a new strategy to study the molecular mechanisms using human lung transplant samples and modern sequencing. The interaction between donor and recipient cells is a unique feature to transplantation. As such, we developed a sequencing and bioinformatic strategy to deconvolute recipient from donor specific gene expression in a frozen lung tissue biopsy. We performed whole exome sequencing (40x coverage) on frozen biopsies of pure donor and recipient lungs, followed by RNA-seq (200M 2x125 bp) on a frozen lung biopsy taken at 2h after reperfusion. For each patient, donor/recipient specific single nucleotide variations (SNVs) were identified and used to quantify donor/recipient expression in the RNA-seq data. The genes were compared to known marker genes from the lung and immune cell types. Recipient-specific transcripts identified from the RNA-seq data were enriched for immune cell specific transcripts and gene ontology analysis depicted neutrophil activation and degranulation (Fig 1). Donor-specific transcripts were enriched in lung parenchymal cell types. Allele fractions of the recipient specific transcripts had a median ∼0.05, while donor specific transcripts had the expected median of 0.5. This indicates that the majority of gene expression is derived from the donor lung and there is only a small fraction of recipient derived transcripts as would be expected in the early reperfusion period. We have demonstrated that it is possible to deconvolute donor and recipient transcripts at the earliest stages of reperfusion by taking advantage of SNVs. The resultant pathway analysis corresponds with our current knowledge of reperfusion biology. Following validation, we aim to apply this to less well-defined phenomena such as CLAD and to generalize the technique to solid-organ transplantation.