Non-invasive Prenatal Diagnosis (NIPD) of Cystic Fibrosis by Quantitative Real Time MEMO-PCR

The recent development of techniques to analyze foetal DNA circulating in blood maternal opened new perspectives for prenatal diagnosis (PD). Non-invasive PND (NIPD) is now the method of choice for the determination of fetal sex (genetic disorders linked to X chromosome) and rhesus genotype. NIPD begins to find applications in the diagnosis of monogenic diseases for mutations of paternal transmission. However a number of technical challenges still hamper diagnostic applications in routine, and have to be resolved taking into account the aspects of cost, reliability (false negative ..) and complexity of equipment and bioinformatics studies.

The investigators propose to develop and validate an analytical and clinical NIPD test for cystic fibrosis from maternal blood by analysis circulating free fetal DNA (cff-DNA), searching for the paternal mutation in families with CFTR compound heterozygosity.

The test method based on MEMO associated with a platform for real-time PCR can be used for the detection of trace DNA mutant. This technique is commonly used for mutations in mosaic (less than 1%) of genes common cancers. Positive detection of the paternal mutation is always checked by a second mini-sequencing technique.

Prior to any specific CFTR test, the DNA profile of each sample will be determined using a commercial kit of Mini STR adapted to study casework. A tri-allelic profile for markers will prove the presence of fetal DNA in the studied specimen and will thus limit false negatives associated with the lack or insufficient amounts of cff-DNA.

The validation step of the investigators analytical methods will be made on chimeric DNA control samples artificially created. Then the test will be clinicaly validated by a retrospective study of maternal serum from pregnant women, who have been the subject of PND or PGD (preimplantation genetic diagnosis) of CF request in the laboratory during the study period (2012 to 2013).