title: Quantification of fetal DNA by use of methylation-based DNA discrimination.
abstract: BACKGROUND
  Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in noninvasive prenatal diagnostics. Most applications rely on the qualitative detection of fetal nucleic acids to determine the genetic makeup of the fetus. This method leads to an analytic dilemma, because test results from samples that do not contain fetal DNA or are contaminated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to maternal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma.
  
  
  METHODS
  The assay used methylation-sensitive restriction enzymes to eliminate the maternal (unmethylated) fraction of the DNA sample. The undigested fetal DNA fraction was then coamplified in the presence of a synthetic oligonucleotide to permit competitive PCR. The amplification products were quantified by single-base extension and MALDI-TOF MS analysis.
  
  
  RESULTS
  Using 2 independent markers, (sex determining region Y)-box 14 (SOX14) and T-box 3 (TBX3), we measured a mean of 151 copies of fetal DNA/mL plasma and a mean fetal fraction of 0.13 in samples obtained from pregnant women. We investigated 242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensitivity and specificity for the assay of 99% and 100%, respectively.
  
  
  CONCLUSIONS
  By investigating several regions in parallel, we reduced the measurement variance and enabled quantification of circulating cell-free DNA. Our results indicate that this multiplex methylation-based reaction detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma.
authors: Sequenom Center for Molecular Medicine, San Diego, CA, USA.
authors: Anders O H
authors: AO
authors: Nygren
authors: Jarrod
authors: J
authors: Dean
authors: Taylor J
authors: TJ
authors: Jensen
authors: Selena
authors: S
authors: Kruse
authors: William
authors: W
authors: Kwong
authors: Dirk
authors: D
authors: van den Boom
authors: Mathias
authors: M
authors: Ehrich
mesh_terms: D004247:DNA / Q000097:blood*; D019175:DNA Methylation; D004262:DNA Restriction Enzymes; D005240:Feasibility Studies; D005260:Female; D005333:Fetus; D005819:Genetic Markers; D015894:Genome, Human; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D010920:Placenta / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011247:Pregnancy / Q000097:blood*; D011296:Prenatal Diagnosis / Q000379:methods; D055749:SOXB2 Transcription Factors / Q000097:blood / Q000235:genetics; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020825:T-Box Domain Proteins / Q000097:blood / Q000235:genetics
chemical_list: D005819:Genetic Markers; C117478:SOX14 protein, human; D055749:SOXB2 Transcription Factors; D020825:T-Box Domain Proteins; C106884:TBX3 protein, human; D004247:DNA; D004262:DNA Restriction Enzymes