title: Use of copy number deletion polymorphisms to assess DNA chimerism.
abstract: BACKGROUND
  We describe a novel approach that harnesses the ubiquity of copy number deletion polymorphisms in human genomes to definitively detect and quantify chimeric DNA in clinical samples. Unlike other molecular approaches to chimerism analysis, the copy number deletion (CND) method targets genomic loci (>50 base pairs in length) that are wholly absent from wild-type (i.e., self) background DNA sequences in a sex-independent manner.
  
  
  METHODS
  Bespoke quantitative PCR (qPCR) CND assays were developed and validated using a series of DNA standards and chimeric plasma DNA samples collected from 2 allogeneic kidney transplant recipients and 12 pregnant women. Assay performance and informativeness were assessed using appropriate statistical methods.
  
  
  RESULTS
  The CND qPCR assays showed high sensitivity, precision, and reliability for linear quantification of DNA chimerism down to 16 genomic equivalents (i.e., 106 pg). Fetal fraction (%) in 12 singleton male pregnancies was calculated using the CND qPCR approach, which showed closer agreement with single-nucleotide polymorphism-based massively parallel sequencing than the SRY (sex determining region Y) (Y chromosome) qPCR assay. The latter consistently underestimated the fetal fraction relative to the other methods. We also were able to measure biological changes in plasma nonself DNA concentrations in 2 renal transplant recipients.
  
  
  CONCLUSIONS
  The CND qPCR technique is suitable for measurement of chimerism for monitoring of rejection in allogeneic organ transplantation and quantification of the cell-free fetal DNA fraction in maternal plasma samples used for noninvasive prenatal genetic testing.
authors.affiliation: Murdoch Childrens Research Institute, Melbourne, VIC, Australia;
authors.forename: Damien L
authors.initials: DL
authors.lastname: Bruno
authors.affiliation: Murdoch Childrens Research Institute, Melbourne, VIC, Australia; Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia;
authors.forename: Devika
authors.initials: D
authors.lastname: Ganesamoorthy
authors.affiliation: Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia; Department of Medical Biology, University of Melbourne, Melbourne, VIC, Australia;
authors.forename: Natalie P
authors.initials: NP
authors.lastname: Thorne
authors.affiliation: Murdoch Childrens Research Institute, Melbourne, VIC, Australia;
authors.forename: Ling
authors.initials: L
authors.lastname: Ling
authors.affiliation: Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia; Department of Mathematics and Statistics, University of Melbourne, Melbourne, VIC, Australia;
authors.forename: Melanie
authors.initials: M
authors.lastname: Bahlo
authors.affiliation: The Australian Genome Research Facility, Parkville, VIC, Australia;
authors.forename: Sue
authors.initials: S
authors.lastname: Forrest
authors.affiliation: Department of Nephrology, Austin Health, Melbourne, VIC, Australia;
authors.forename: Marieke
authors.initials: M
authors.lastname: Veenendaal
authors.affiliation: Department of Nephrology, Austin Health, Melbourne, VIC, Australia;
authors.forename: Marina
authors.initials: M
authors.lastname: Katerelos
authors.affiliation: Department of Anatomical Pathology, Austin Hospital, Melbourne, VIC, Australia.
authors.forename: Alison
authors.initials: A
authors.lastname: Skene
authors.affiliation: Department of Nephrology, Austin Health, Melbourne, VIC, Australia;
authors.forename: Frank L
authors.initials: FL
authors.lastname: Ierino
authors.affiliation: Department of Nephrology, Austin Health, Melbourne, VIC, Australia;
authors.forename: David A
authors.initials: DA
authors.lastname: Power
authors.affiliation: Murdoch Childrens Research Institute, Melbourne, VIC, Australia; Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia; howard.slater@vcgs.org.au.
authors.forename: Howard R
authors.initials: HR
authors.lastname: Slater
mesh_terms: D002678:Chimera / Q000235:genetics*; D056915:DNA Copy Number Variations; D006801:Humans; D057230:Limit of Detection; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results