title: Non-invasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control.
abstract: BACKGROUND
  Analysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA.
  
  
  METHODS
  To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome.
  
  
  RESULTS
  The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit.
  
  
  CONCLUSIONS
  This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.
authors.forename: Claire
authors.initials: C
authors.lastname: Guissart
authors.forename: Vanessa
authors.initials: V
authors.lastname: Debant
authors.forename: Marie
authors.initials: M
authors.lastname: Desgeorges
authors.forename: Corinne
authors.initials: C
authors.lastname: Bareil
authors.forename: Caroline
authors.initials: C
authors.lastname: Raynal
authors.forename: Caroline
authors.initials: C
authors.lastname: Toga
authors.forename: Victoria
authors.initials: V
authors.lastname: Pritchard
authors.forename: Michel
authors.initials: M
authors.lastname: Koenig
authors.forename: Mireille
authors.initials: M
authors.lastname: Claustres
authors.forename: Marie-Claire
authors.initials: MC
authors.lastname: Vincent
mesh_terms: D003550:Cystic Fibrosis / Q000175:diagnosis / Q000235:genetics*; D005260:Female; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D011247:Pregnancy