Non-medical applications of non invasive prenatal testing: ethical issues and apllicabilities

The possibility of obtaining material for foetal molecular analysis without the need of invasive procedures has been a long wished improvement of practice in prenatal diagnostics. The demonstration of the presence of foetal cells and circulating foetal free-DNA in a sample of mother-to-be’s blood promised that a non-invasive approach for prenatal diagnostics is near to becoming a reality. \nThe presence of foetal cells (albeit in low numbers) in maternal blood has been known since 1893, when Schomorl [1] described throphoblast cells in lung circulation of pregnant women who deceased from eclampsia. However, following the first observation numerous attempts to isolate these cells have proven disappointing. The main reasons for the lack of success with fetal cells isolation can be attributed to the very tiny proportion of fetal cells in the total maternal blood cell population. Moreover, isolation of fetal cells failed to demonstrate the origin of these cells using genetic profiling. \nThe presence of cell-free foetal circulating DNA sequences in the plasma of pregnant women was first described in 1997, when the Lo group [2] reported the presence of Y chromosome DNA sequences in pregnant circulation. \nThe demonstration of foetal genetic material in maternal circulation incited a new era of non-invasive prenatal diagnostics based on free foetal DNA, with the purpose of replacing the invasive approaches based on villi sampling, cordonocentesis and amniocentesis. Obtaining chorionic villi, amniocentesis or cord blood specimens is expensive and requires an invasive procedure that carries a small risk to the foetus and the possibility of adverse maternal effects, such as unnerving risk of miscarriage. Moreover, these tests are not provided until late in the first trimester thus implying a rate – limiting step in the provision of prenatal diagnosis: only pregnant women at the highest risk of having a foetus with a genetic syndrome or another disorder of major clinical significance have been eligible to these tests. \nIn this study we considered that genetic prenatal diagnosis could have forensic applications, in particular in cases of rape resulting in pregnancy. So, we explored the opportunity and ethical issues related to the introduction of non-invasive prenatal testing for non-medical applications, such as sex determination and paternity testing. In particular, based on our laboratory analyses we achieved proof of principle of a non-invasive test for forensic purposes. \nWe obtain approval for our studies from the Ethics Committee of the University - Hospital of Padova (Protocol n. 2105 P / 2010), Italy. First of all we demonstrated the applicability of cell free DNA in human identification. Then, we enrolled pregnant women and their partner and investigated the STR profiles obtained from free DNA and nucleated red blood cells in peripheral blood of pregnant women that were compared to profiles of the putative father, in order to define paternity. The STR profiles in free DNA were investigated using consolidated methods, by performing multiplex PCR and sequencing, without succesfull results that we attributed to the low sensitivity of the method. This result pushed us to investigate the applicability of new high throughput technologies (e.g. next generation sequencing) in plasma DNA of mother’s-to be. This new approach, of potential interest for forensic genetics, seems to be promizing thank to its high sensitivity. \nThe best results with profiling fetal DNA from maternal blood specimens were obtained from DNA profiling of nucleated red blood cells of embryonic origin when enrichment by Fluorescent Activated Cell Sorting (FACS) was performed. For enrichment a combination of monoclonal antibodies was employed: all amplified alleles amplified from sorted cells, others than those of the mother, matched the alleles of the putative father. \nOur study, which is the first one to investigate DNA profiling analysis on nucleated fetal red blood cells for forensic genetics, demonstrated that non-invasive prenatal paternity testing could be developed as a tool for the identification of perpetrators of rape in case of resulting pregnancy. More efforts need to be spend to optimize the isolation of fetal cells. The proof of principle that non invasive prenatal paternity testing on DNA of rare fetal cells circulating in pregnant blood is feasible justifies further efforts in developing this new approach. \n