title: A Non-Invasive Droplet Digital PCR (ddPCR) Assay to Detect Paternal CFTR Mutations in the Cell-Free Fetal DNA (cffDNA) of Three Pregnancies at Risk of Cystic Fibrosis via Compound Heterozygosity.
abstract: BACKGROUND
  Non-invasive prenatal diagnosis (NIPD) makes use of cell-free fetal DNA (cffDNA) in the mother's bloodstream as an alternative to invasive sampling methods such as amniocentesis or CVS, which carry a 0.5-1% risk of fetal loss. We describe a droplet digital PCR (ddPCR) assay designed to inform the testing options for couples whose offspring are at risk of suffering from cystic fibrosis via compound heterozygosity. By detecting the presence or absence of the paternal mutation in the cffDNA, it is possible to predict whether the fetus will be an unaffected carrier (absence) or whether further invasive testing is indicated (presence).
  
  
  METHODS
  We selected a family in which the parents were known to carry different mutated CFTR alleles as our test system. NIPD was performed for three of their pregnancies during the first trimester (at around 11-12 weeks of gestation). Taqman probes were designed against an amplicon in exon 11 of the CFTR gene, to quantify the proportion of mutant (ΔF508-MUT; FAM) and normal (ΔF508-NOR; VIC) alleles at position c.1521_1523 of the CFTR gene.
  
  
  CONCLUSIONS
  The assay correctly and unambiguously recognized the ΔF508-MUT CFTR allele in the cffDNA of all three proband fetuses and none of the six unaffected control fetuses. In conclusion, the Bio-Rad QX100 was found to be a cost-effective and technically undemanding platform for designing bespoke NIPD assays.
authors.affiliation: West Midlands Regional Genetics Laboratory, Birmingham Women's NHS Foundation Trust, Mindelsohn Way, Edgbaston, Birmingham, B15 2TG, United Kingdom.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Debrand
authors.affiliation: West Midlands Regional Genetics Laboratory, Birmingham Women's NHS Foundation Trust, Mindelsohn Way, Edgbaston, Birmingham, B15 2TG, United Kingdom.|Department of Medical Genetics, Athens University, Agia Sofia Children's Hospital, 11527, Athens, Greece.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Lykoudi
authors.affiliation: West Midlands Regional Genetics Laboratory, Birmingham Women's NHS Foundation Trust, Mindelsohn Way, Edgbaston, Birmingham, B15 2TG, United Kingdom.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Bradshaw
authors.affiliation: West Midlands Regional Genetics Laboratory, Birmingham Women's NHS Foundation Trust, Mindelsohn Way, Edgbaston, Birmingham, B15 2TG, United Kingdom.
authors.forename: Stephanie K
authors.initials: SK
authors.lastname: Allen
mesh_terms: D000483:Alleles; D002474:Cell-Free System; D003550:Cystic Fibrosis / Q000175:diagnosis* / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D005192:Family Health; D005220:Fathers; D005260:Female; D005838:Genotype; D006579:Heterozygote; D006801:Humans; D008297:Male; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011261:Pregnancy Trimester, First; D011296:Prenatal Diagnosis / Q000379:methods*; D012307:Risk Factors
chemical_list: C505032:CFTR protein, human; D017931:DNA Primers; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator