Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations that frequently concern prenatal diagnosis requests. The objective of this study was to develop an approach to isolate and analyze Circulating Trophoblastic Fetal Cells (CFTCs) for NIPD of monogenic diseases caused by triplet repeat expansion or point mutations. We developed a method for CFTC isolation based on DEPArray sorting and used Huntington's disease as the clinical model for CFTC-based NIPD. Then, we investigated whether CFTC isolation and Whole Genome Amplification (WGA) could be used for NIPD in couples at risk of transmitting different monogenic diseases. Our data show that the allele drop-out rate was 3-fold higher in CFTCs than in maternal cells processed in the same way. Moreover, we give new insights into CFTCs by compiling data obtained by extensive molecular testing by microsatellite multiplex PCR genotyping and by WGA followed by mini-exome sequencing. CFTCs appear to be often characterized by a random state of genomic degradation. Non-Invasive prenatal diagnosis (NIPD) of monogenic diseases, based on the analysis of circulating cell-free fetal DNA (cff-DNA) 1-3 , is a safer alternative to invasive prenatal testing methods (amniocentesis and choriocentesis) that entail a significant risk of miscarriage (0.5-1%) 4. However, technical issues related to cff-DNA characteristics remain 5. Consequently, not all mutations can be investigated with this approach, particularly triplet expansion mutations that concern rare and incurable diseases (e.g., Steinert myotonic dystrophy, Huntington's disease,

The detection of two frequent CFTR disease-causing variations in the context of a newborn screening program (NBS) usually leads to the diagnosis of cystic fibrosis (CF) and a relevant genetic counseling in the family. In the present study, CF-causing variants p.Phe508del (F508del) and c.3140-26A>G (3272-26A>G) were identified on a neonate with positive ImmunoReactive Trypsinogen test by the Elucigene™ CF30 kit. The CF diagnosis initially suggested, despite three inconclusive Sweat Chloride Tests (SCT), was finally ruled out after the familial segregation study combined with a negative SCT. Haplotype studies, based on the comparison of 80 p.Phe508del haplotypes, suggested a probable de novo occurrence of c.3140-26A>G on the p.Phe508del ancestral allele in this family. This false positive case emphasizes the importance of SCT in the NBS strategy. Moreover, it raises the need for familial segregation studies in CF and in overall molecular diagnosis strategy of autosomal recessive diseases.

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.

We describe one of the largest studies evaluating the Ion Proton based NIPS and the first clinical study reporting pregnancy outcome in a large set of patients. We demonstrate that this platform is highly efficient in detecting the three most common trisomies. Our protocol is robust and can be easily implemented in any medical genetics laboratory. This article is protected by copyright. All rights reserved.

Background: Analysis of cell-free fetal DNA in maternal plasma is very promising for early diagnosis of monogenic diseases. However, it has been limited by the need to set up patient- or disease-specific custom-made approaches. Here we propose a universal test based on fluorescent multiplex PCR and size fragment analysis for an indirect diagnosis of cystic fibrosis (CF). Methods: The test, based on haplotyping, includes nine intra- and extragenic short tandem repeats of the CFTR locus, the coamplification of p.Phe508del (the most frequent mutation in CF patients worldwide), and a specific SRY sequence. The assay is able to determine the inherited paternal allele. Results: Our simple approach was successfully applied to 30 couples and provided clear results from the maternal plasma. The mean rate of informative markers was sufficient to propose it for use in indirect diagnosis. Conclusions: This noninvasive prenatal diagnosis test, focused on indirect diagnosis of CF, offers many advantages over current methods: it is simple, rapid, and cost-effective. It allows for the testing of a large number of couples with high risk of CF, whatever the familial mutation of the CFTR gene. It provides an alternative method to reduce the number of invasive tests.