title: An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping.
abstract: Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources (i.e., donor and recipient tissues after transplantation) is challenging. In human cellular transplantation, there is currently no useable method to detectin vivoengraftment, and blood-based non-invasive tests for allograft rejection in solid organ transplantation are either non-specific or absent. Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm.In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. Forin vivotesting, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation.
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary , Calgary, AB , Canada.
authors.forename: Paul M K
authors.initials: PM
authors.lastname: Gordon
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Paediatrics, University of Calgary, Calgary, AB, Canada; Department of Medical Genetics, University of Calgary, Calgary, AB, Canada.
authors.forename: Aneal
authors.initials: A
authors.lastname: Khan
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Paediatrics, University of Calgary, Calgary, AB, Canada.
authors.forename: Umair
authors.initials: U
authors.lastname: Sajid
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Paediatrics, University of Calgary, Calgary, AB, Canada.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Chang
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Paediatrics, University of Calgary, Calgary, AB, Canada.
authors.forename: Varun
authors.initials: V
authors.lastname: Suresh
authors.affiliation: Molecular Diagnostic Laboratory, Genetic Laboratory Services-South, Alberta Health Services , Calgary, AB , Canada.
authors.forename: Leo
authors.initials: L
authors.lastname: Dimnik
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Medical Genetics, University of Calgary, Calgary, AB, Canada; Molecular Diagnostic Laboratory, Genetic Laboratory Services-South, Alberta Health Services, Calgary, AB, Canada.
authors.forename: Ryan E
authors.initials: RE
authors.lastname: Lamont
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Medical Genetics, University of Calgary, Calgary, AB, Canada; Molecular Diagnostic Laboratory, Genetic Laboratory Services-South, Alberta Health Services, Calgary, AB, Canada.
authors.forename: Jillian S
authors.initials: JS
authors.lastname: Parboosingh
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Paediatrics, University of Calgary, Calgary, AB, Canada.
authors.forename: Steven R
authors.initials: SR
authors.lastname: Martin
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Richard T
authors.initials: RT
authors.lastname: Pon
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary , Calgary, AB , Canada.
authors.forename: Jene
authors.initials: J
authors.lastname: Weatherhead
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary , Calgary, AB , Canada.
authors.forename: Shelly
authors.initials: S
authors.lastname: Wegener
authors.affiliation: Department of Cardiac Sciences, Libin Cardiovascular Institute of Alberta, University of Calgary , Calgary, AB , Canada.
authors.forename: Debra
authors.initials: D
authors.lastname: Isaac
authors.affiliation: Alberta Children's Hospital Research Institute, University of Calgary, Calgary, AB, Canada; Department of Paediatrics, University of Calgary, Calgary, AB, Canada; Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, AB, Canada; Department of Cardiac Sciences, Libin Cardiovascular Institute of Alberta, University of Calgary, Calgary, AB, Canada.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Greenway
keywords: biomarker; cell-free DNA; heart; hepatocyte transplantation; solid organ transplantation; transplantation