Diagnosi prenatale non invasiva di malattie monogeniche attraverso la ricerca e l'isolamento di cellule e DNA fetale nel sangue materno

Prenatal genetic diagnosis of monogenic diseases and chromosomal abnormalities is usually performed collecting fetal samples through villocentesis or amniocentesis. These invasive procedures are associated with 0.5-1% risk for the fetus. Due to it, in recent years, much effort has been made to develop non invasive prenatal diagnosis (NIPD). Two potential non invasive approaches involve the analysis of fetal cells and cell-free fetal DNA (cffDNA) found in the maternal circulation. The presence of fetal cells in the maternal circulation was first documented by 1893 when the fetal cells were found in pregnant women died for eclampsia. The Fetal erythroblasts (NRBC) from the maternal circulation have been considered the best potential target for NIPD because they can be detected early in pregnancy, are nucleated and have short life. The NRBC isolation from maternal circulation is still difficult because the number of NRBC is small and a fetal specific antibody is not currently available. Cff-DNA detected in the maternal circulation during pregnancy, could potentially offer an excellent method for early NIPD of the genetic status of a fetus. In a recent study has been shown that, during the pregnancy, the cffDNA is cleared from the maternal circulation, 16 min following delivery. Moreover, it has been demonstrated that cffDNA has a mean fractional concentration of about 10%. The goal of our project was to develop and validate a protocol for NIPD of monogenic diseases by two experimental procedures: the isolation and the molecular characterization of fetal NRBC, and the analysis of cffDNA in maternal plasma. We recruited 95 pregnant women between 6th and 14th week of gestation. In our study we have developed a protocol for fetal NRBC isolation from maternal blood that unfortunately require further refinements the isolation procedure. In parallel we have developed a protocol for non invasive fetal sexing from cffDNA. This method appears to be highly accurate showing 100% sensitivity and 96% specificity. In the next future we are planning to analyze cffDNA both for monogenic diseases and for the most frequent aneuploidies using massively parallel sequencing.