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.

Lucie Orhant (1) , Olivia Anselem (2) , Mélanie Fradin (3) , Pierre Hadrien Becker (1) , Caroline Beugnet (1) , Nathalie Deburgrave (1) , Gilles Tafuri (1) , Franck Letourneur (4) , François Goffinet (2) , Laïla Allach El Khattabi (5) , France Leturcq (1) , Thierry Bienvenu (1, 4) , Vassilis Tsatsaris (2) , Juliette Nectoux (1, 4)

1 Biochimie et biologie moléculaire
2 Service de Gynécologie et Obstétrique [Cochin]
3 IGDR - Institut de Génétique et Développement de Rennes
4 IC UM3 (UMR 8104 / U1016) - Institut Cochin
5 Laboratoire de cytogénétique constitutionnelle pré et post natale

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.

Domains: Life Sciences [q-bio]

Lucie Orhant, Olivia Anselem, Mélanie Fradin, Pierre Hadrien Becker, Caroline Beugnet, et al.. 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.. Prenatal Diagnosis, 2016, 36 (5), pp.397-406. ⟨10.1002/pd.4790⟩. ⟨hal-01272602⟩