Use of copy number variation polymorphism to assess cellular and cell-free DNA chimerism following transplantation

A state of genetic chimerism is created by allogenic transplantation, the quality and quantity of which may have diagnostic significance. The monitoring of cellular macrochimerism following allogeneic hematopoietic cell transplantation (HCT) is standard of care. It provides an assessment of engraftment, prognostic information, and guides therapeutic interventions. Separately, the discovery of cell-free DNA (cfDNA) chimerism following solid organ transplantation has raised the possibility that increases in donor genotype graft-derived cfDNA may be biomarker for rejection. As a result of these and other developments, the clinical and research requirements for chimerism analysis in the transplantation context exceeds the capabilities offered by existing methodologies. To address this, a panel of 39 droplet digital PCR (ddPCR) assays was developed. These targeted highly polymorphic copy number variation (CNV) loci strategically selected using a population genomics approach to maximise the probability of a donor one copy, recipient zero copy genotype combination (and vice versa). These informative combinations create a negative background against which absolute quantification of the one copy genotype can be performed using ddPCR. Analytical validation of the developed chimerism analysis method for absolute quantification of cfDNA was performed. All studied assays performed linearly across the range <6-1280 copies/mL. Limit of blank was 0 copies/mL, limit of detection was 6 copies/mL, and limit of quantification was 8 copies/mL. A method for genotyping donor and recipient informative CNV loci from chimeric cfDNA was developed and conceptually validated using donor and recipient genomic DNA. The developed chimerism analysis method was used to evaluate the diagnostic validity of absolute measurements of plasma graft-derived cfDNA and total cfDNA, as well as the relative measure of graft fraction, for the diagnosis of kidney transplant (KT) rejection. 61 indication biopsy-ma