Clin Chem Lab Med 2018; 56(5): 728–738 Aurélia Gruber a , Mathilde Pacault a , Laila Allach El Khattabi, Nicolas Vaucouleur, Lucie Orhant, Thierry Bienvenu, Emmanuelle Girodon, Dominique Vidaud, France Leturcq, Catherine Costa, Franck Letourneur, Olivia Anselem, Vassilis Tsatsaris, François Goffinet, Géraldine Viot, Michel Vidaud and Juliette Nectoux* Non-invasive prenatal diagnosis of paternally inherited disorders from maternal plasma: detection of NF1 and CFTR mutations using droplet digital PCR https://doi.org/10.1515/cclm-2017-0689 Received August 3, 2017; accepted November 23, 2017 Abstract Background: To limit risks of miscarriages associated with invasive procedures of current prenatal diagnosis prac- tice, we aim to develop a personalized medicine-based protocol for non-invasive prenatal diagnosis (NIPD) of monogenic disorders relying on the detection of pater- nally inherited mutations in maternal blood using droplet digital PCR (ddPCR). Methods: This study included four couples at risk of transmitting paternal neurofibromatosis type 1 (NF1) mutations and four couples at risk of transmitting com- pound heterozygous CFTR mutations. NIPD was per- formed between 8 and 15 weeks of gestation, in parallel to conventional invasive diagnosis. We designed specific hydrolysis probes to detect the paternal mutation and to assess the presence of cell-free fetal DNA by ddPCR. Ana- lytical performances of each assay were determined from paternal sample, an then fetal genotype was inferred from maternal plasma sample. Results: Presence or absence of the paternal mutant allele was correctly determined in all the studied plasma DNA samples. Conclusions: We report an NIPD protocol suitable for implementation in an experienced laboratory of molecu- lar genetics. Our proof-of-principle results point out a high accuracy for early detection of paternal NF1 and CFTR mutations in cell-free DNA, and open new perspectives for extending the technology to NIPD of many other mono- genic diseases. Keywords: cystic fibrosis; droplet digital PCR; neuro- fibromatosis type 1; non-invasive prenatal diagnosis; paternal mutation; personalized medicine. Introduction The presence of fetal DNA in pregnant women plasma [1] allowed the development of non-invasive prenatal testing for aneuploidies and non-invasive prenatal diagnosis (NIPD) for severe monogenic diseases through maternal blood collection, thus eliminating the fetal loss risk due to invasive procedures [2]. Cell-free fetal DNA (cffDNA) coex- ists in maternal plasma with a large background of mater- nal cell-free DNA (cfDNA) predominantly derived from the mother’s blood cells [3, 4]. Thus, one way to reliably detect fetal DNA in maternal plasma is to analyze nucleotidic sequences that are specific to the fetus and distinguish- able from the maternal DNA, such as paternally inherited mutations. In this context, digital PCR represents a highly sensi- tive and reproducible method that could serve as an appro- priate tool for fetal mutational status analysis. In contrast to real-time quantitative PCR performed in one single reaction volume, droplet digital PCR (ddPCR) allows for the partitioning of the sample into thousands of nanoliter droplets, each constituting an individual nanoreactor. Each droplet contains either zero or one copy of the target DNA, following a Poisson distribution [5, 6]. After allele- specific PCR reaction in presence of fluorogenic probes, a Aurélia Gruber and Mathilde Pacault contributed equally to this work. *Corresponding author: Juliette Nectoux, Service de Génétique et Biologie Moléculaires, HUPC Hôpital Cochin, 27 rue du Faubourg Saint Jacques, 75014 Paris, France, Phone: 00 33 1 58 41 16 22, Fax: 00 33 1 58 41 15 80, E-mail: juliette.nectoux@aphp.fr. http://orcid.org/0000-0001-5584-1112 Aurélia Gruber, Mathilde Pacault, Nicolas Vaucouleur, Lucie Orhant, Thierry Bienvenu, Emmanuelle Girodon, Dominique Vidaud, France Leturcq, Catherine Costa and Michel Vidaud: Service de Génétique et Biologie Moléculaires, HUPC Hôpital Cochin, Paris, France Laila Allach El Khattabi: Service de Cytogénétique, HUPC Hôpital Cochin, Paris, France Franck Letourneur: INSERM, U1016, Institut Cochin, CNRS UMR8104, Université Paris Descartes, Paris, France Olivia Anselem, Vassilis Tsatsaris, François Goffinet and Géraldine Viot: Maternité Cochin-Port Royal, HUPC Hôpital Cochin, Paris, France Brought to you by | Umea University Library - invalid, see 15197 - Authenticated Download Date | 4/6/18 10:59 AM