Droplet digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia

Prenat Diagn. 2016 May;36(5):397-406. doi: 10.1002/pd.4790. Epub 2016 Apr 7.

Abstract

Background: Achondroplasia is generally detected by abnormal prenatal ultrasound findings in the third trimester of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations.

Methods: To determine whether the fetus carries the de novo mis-sense genetic mutation at nucleotide 1138 in FGFR3 gene involved in >99% of achondroplasia cases, we developed two independent methods: digital-droplet PCR combined with minisequencing, which are very sensitive methods allowing detection of rare alleles.

Results: We collected 26 plasmatic samples from women carrying fetus at risk of achondroplasia and diagnosed to date a total of five affected fetuses in maternal blood. The sensitivity and specificity of our test are respectively 100% [95% confidence interval, 56.6-100%] and 100% [95% confidence interval, 84.5-100%].

Conclusions: This novel, original strategy for non-invasive prenatal diagnosis of achondroplasia is suitable for implementation in routine clinical testing and allows considering extending the applications of these technologies in non-invasive prenatal diagnosis of many other monogenic diseases. © 2016 John Wiley & Sons, Ltd.

MeSH terms

  • Achondroplasia / blood
  • Achondroplasia / diagnosis*
  • Achondroplasia / genetics
  • Algorithms
  • Case-Control Studies
  • DNA / blood*
  • DNA / genetics
  • Female
  • Humans
  • Maternal Serum Screening Tests*
  • Mutation, Missense
  • Polymerase Chain Reaction
  • Pregnancy
  • Prenatal Diagnosis
  • Receptor, Fibroblast Growth Factor, Type 3 / genetics
  • Sensitivity and Specificity
  • Sequence Analysis, DNA

Substances

  • DNA
  • FGFR3 protein, human
  • Receptor, Fibroblast Growth Factor, Type 3