title: Mrassf1a-pap, a novel methylation-based assay for the detection of cell-free fetal DNA in maternal plasma.
abstract: OBJECTIVE
  RASSF1A has been described to be differentially methylated between fetal and maternal DNA and can therefore be used as a universal sex-independent marker to confirm the presence of fetal sequences in maternal plasma. However, this requires highly sensitive methods. We have previously shown that Pyrophosphorolysis-activated Polymerization (PAP) is a highly sensitive technique that can be used in noninvasive prenatal diagnosis. In this study, we have used PAP in combination with bisulfite conversion to develop a new universal methylation-based assay for the detection of fetal methylated RASSF1A sequences in maternal plasma.
  
  
  METHODS
  Bisulfite sequencing was performed on maternal genomic (g)DNA and fetal gDNA from chorionic villi to determine differentially methylated regions in the RASSF1A gene using bisulfite specific PCR primers. Methylation specific primers for PAP were designed for the detection of fetal methylated RASSF1A sequences after bisulfite conversion and validated.
  
  
  RESULTS
  Serial dilutions of fetal gDNA in a background of maternal gDNA show a relative percentage of ~3% can be detected using this assay. Furthermore, fetal methylated RASSF1A sequences were detected both retrospectively as well as prospectively in all maternal plasma samples tested (n = 71). No methylated RASSF1A specific bands were observed in corresponding maternal gDNA. Specificity was further determined by testing anonymized plasma from non-pregnant females (n = 24) and males (n = 21). Also, no methylated RASSF1A sequences were detected here, showing this assay is very specific for methylated fetal DNA. Combining all samples and controls, we obtain an overall sensitivity and specificity of 100% (95% CI 98.4%-100%).
  
  
  CONCLUSIONS
  Our data demonstrate that using a combination of bisulfite conversion and PAP fetal methylated RASSF1A sequences can be detected with extreme sensitivity in a universal and sex-independent manner. Therefore, this assay could be of great value as an addition to current techniques used in noninvasive prenatal diagnostics.
authors.affiliation: Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Jessica M E
authors.initials: JM
authors.lastname: van den Oever
authors.affiliation: Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Sahila
authors.initials: S
authors.lastname: Balkassmi
authors.affiliation: Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Tim
authors.initials: T
authors.lastname: Segboer
authors.affiliation: Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: E Joanne
authors.initials: EJ
authors.lastname: Verweij
authors.affiliation: Department of Ophthalmology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Pieter A
authors.initials: PA
authors.lastname: van der Velden
authors.affiliation: Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Dick
authors.initials: D
authors.lastname: Oepkes
authors.affiliation: Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Egbert
authors.initials: E
authors.lastname: Bakker
authors.affiliation: Department of Clinical Genetics, Laboratory for Diagnostic Genome Analysis (LDGA), Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Elles M J
authors.initials: EM
authors.lastname: Boon
mesh_terms: D001483:Base Sequence; D002474:Cell-Free System; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification; D019175:DNA Methylation; D005260:Female; D005333:Fetus / Q000378:metabolism*; D005819:Genetic Markers / Q000235:genetics*; D005838:Genotype; D005865:Gestational Age; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010920:Placenta / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D011446:Prospective Studies; D012189:Retrospective Studies; D012689:Sequence Homology, Nucleic Acid; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: D005819:Genetic Markers; C411762:RASSF1 protein, human; D025521:Tumor Suppressor Proteins; D004247:DNA