title: Non-invasive prenatal diagnosis (NIPD) of cystic fibrosis: an optimized protocol using MEMO fluorescent PCR to detect the p.Phe508del mutation.
abstract: BACKGROUND
  Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches.
  
  
  METHODS
  To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence.
  
  
  RESULTS
  We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses.
  
  
  CONCLUSIONS
  This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France.
authors.forename: C
authors.initials: C
authors.lastname: Guissart
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France.
authors.forename: C
authors.initials: C
authors.lastname: Dubucs
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France.
authors.forename: C
authors.initials: C
authors.lastname: Raynal
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France.
authors.forename: A
authors.initials: A
authors.lastname: Girardet
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France.
authors.forename: F
authors.initials: F
authors.lastname: Tran Mau Them
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France.
authors.forename: V
authors.initials: V
authors.lastname: Debant
authors.affiliation: CHU de Nice, Service de Génétique Médicale, Nice, France.
authors.forename: C
authors.initials: C
authors.lastname: Rouzier
authors.affiliation: CHU de Nice, Service de Génétique Médicale, Nice, France.
authors.forename: A
authors.initials: A
authors.lastname: Boureau-Wirth
authors.affiliation: CHU de Montpellier, Service de Génétique Médicale, Montpellier, France.
authors.forename: E
authors.initials: E
authors.lastname: Haquet
authors.affiliation: CHU de Montpellier, Service de Génétique Médicale, Montpellier, France.
authors.forename: J
authors.initials: J
authors.lastname: Puechberty
authors.affiliation: CHU de Toulouse, Service de Génétique Médicale, Toulouse, France.
authors.forename: E
authors.initials: E
authors.lastname: Bieth
authors.affiliation: CHU de Toulouse, Service de Génétique Médicale, Toulouse, France.
authors.forename: D
authors.initials: D
authors.lastname: Dupin Deguine
authors.affiliation: CHU de Nîmes, Laboratoire de Cytologie Clinique et Cytogénétique, Nîmes, France.
authors.forename: P
authors.initials: P
authors.lastname: Khau Van Kien
authors.affiliation: Hôpital Saint-Joseph, Service de Diagnostic Prénatal, Marseille, France.
authors.forename: M P
authors.initials: MP
authors.lastname: Brechard
authors.affiliation: Brammer Bio, Alachua, FL, USA.
authors.forename: V
authors.initials: V
authors.lastname: Pritchard
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France.
authors.forename: M
authors.initials: M
authors.lastname: Koenig
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France.
authors.forename: M
authors.initials: M
authors.lastname: Claustres
authors.affiliation: Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France. Electronic address: marie-claire.vincent@inserm.fr.
authors.forename: M C
authors.initials: MC
authors.lastname: Vincent
mesh_terms: D000328:Adult; D000649:Amniocentesis / Q000379:methods*; D015193:Chorionic Villi Sampling / Q000379:methods*; D057186:Comparative Effectiveness Research; D003550:Cystic Fibrosis / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods; D005260:Female; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods; D015203:Reproducibility of Results
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator
keywords: Cystic fibrosis; MEMO-PCR; NIPD; Non-invasive prenatal diagnosis; cff-DNA; p.Phe508del