title: Non-invasive prenatal diagnosis of beta-thalassemia and sickle-cell disease using pyrophosphorolysis-activated polymerization and melting curve analysis.
abstract: OBJECTIVE
  The aim of this study was to develop a pyrophosphorolysis-activated polymerization (PAP) assay for non-invasive prenatal diagnosis (NIPD) of β-thalassemia major and sickle-cell disease (SCD). PAP is able to detect mutations in free fetal DNA in a highly contaminating environment of maternal plasma DNA.
  
  
  METHODS
  Pyrophosphorolysis-activated polymerization primers were designed for 12 informative SNPs, genotyped by melting curve analysis (MCA) in both parents. The PAP assay was tested in a series of 13 plasma DNA samples collected from pregnant women. A retrospective NIPD was performed in a couple at risk for SCD.
  
  
  RESULTS
  All PAP reactions were optimized and able to detect <3% target gDNA in a background of >97% wildtype gDNA. In all 13 cases, the paternal allele was detected by PAP in maternal plasma at 10 to 18 weeks of gestation. For the couple at risk, PAP showed presence of the normal paternal SNP allele in maternal plasma, which was confirmed by results of the chorionic villus sampling analysis.
  
  
  CONCLUSIONS
  In contrast to other methods used for NIPD, the combined PAP and MCA analysis detecting the normal paternal allele is also applicable for couples at risk carrying the same mutation, provided that a previously born child is available for testing to determine the linkage to the paternal SNPs.
authors.affiliation: Hemoglobinopathies Laboratory, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands. m.phylipsen@lumc.nl
authors.forename: Marion
authors.initials: M
authors.lastname: Phylipsen
authors.forename: Supawadee
authors.initials: S
authors.lastname: Yamsri
authors.forename: Emmely E
authors.initials: EE
authors.lastname: Treffers
authors.forename: Diahann T S L
authors.initials: DT
authors.lastname: Jansen
authors.forename: Warsha A
authors.initials: WA
authors.lastname: Kanhai
authors.forename: Elles M J
authors.initials: EM
authors.lastname: Boon
authors.forename: Piero C
authors.initials: PC
authors.lastname: Giordano
authors.forename: Supan
authors.initials: S
authors.lastname: Fucharoen
authors.forename: Egbert
authors.initials: E
authors.lastname: Bakker
authors.forename: Cornelis L
authors.initials: CL
authors.lastname: Harteveld
mesh_terms: D000483:Alleles; D000755:Anemia, Sickle Cell / Q000175:diagnosis*; D004247:DNA / Q000097:blood; D005220:Fathers; D005260:Female; D006580:Genetic Carrier Screening; D008040:Genetic Linkage; D060005:Genotyping Techniques; D006801:Humans; D007962:Leukocytes / Q000737:chemistry; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction; D058105:Polymerization; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D055544:beta-Globins / Q000235:genetics; D017086:beta-Thalassemia / Q000175:diagnosis*
chemical_list: D055544:beta-Globins; D004247:DNA