The Development and Validation of a Novel Non-Invasive Assay Based on Cell Free-DNA to Detect Acute Allograft Rejection After Heart Transplantation
Immune-mediated injury (rejection) of a transplanted organ is a serious problem that can lead to allograft dysfunction and patient death. The gold standard for diagnosing acute cellular rejection (ACR) after heart transplantation (HT) is the endomyocardial biopsy (EMB), an invasive procedure with significant limitations. Dying cells release fragments of DNA into the circulation and increased levels of donor-derived cell-free DNA (dd-cfDNA) have been associated with ACR. Current methods to measure dd-cfDNA employ single nucleotide polymorphisms (SNPs) but an epigenetics-based assay could also accurately quantify dd-cfDNA in recipient blood. This thesis aimed to validate the use of ventricle-specific methylation patterns in human cfDNA as an alternative and novel biomarker for ACR following HT. We hypothesized that dd-cfDNA released due to ACR-mediated injury could be quantified in recipient plasma based on epigenetic differences and would correlate better with tissue apoptosis than EMB-based rejection. We identified increased cellular apoptosis within the myocardium as the severity of ACR increased, which provided a biological rationale for the use of cfDNA as a biomarker for rejection. We also initiated validation of an alternative sequencing platform and panel of highly polymorphic SNPs, which may improve a previously-established SNP-based assay. Finally, we established a bioinformatic pipeline for the identification of ventricle-specific differentially methylated regions (DMRs). These DMRs underwent retrospective validation using cfDNA samples from adult HT patients, which were associated with a known biopsy-proven rejection grade, to demonstrate the ability of these novel blood biomarkers to non-invasively detect acute rejection following HT. In conclusion, we successfully demonstrated the efficacy of cfDNA as a biomarker for immune-mediated tissue injury and introduced the potential use of two ventricle-specific DMRs for the identification and quantification of cfDNA released from a donated heart due to ACR.
heart transplantation, endomyocardial biopsy, acute cellular rejection, non-invasive, cell-free DNA, dd-cfDNA, differentially methylated regions, DMRs, ventricle-specific
Pattar, S. K. (2019). The Development and Validation of a Novel Non-Invasive Assay Based on Cell Free-DNA to Detect Acute Allograft Rejection After Heart Transplantation (Unpublished master's thesis). University of Calgary, Calgary, AB.