Cell-based non-invasive prenatal diagnosis

Genetic prenatal diagnosis is dependent on procurement of fetal cells. This is done my invasive methods such as amniocentesis and chorionic villous sampling. These methods are associated to a risk of 0.5 - 1% of subsequent abortus. For this reason genetic prenatal diagnosis is restricted to pregnancies with are associated with an increased risk of renetik disease of the fetus, such as age of the pregnant women higher than 35 years. In fact the majority of children with genetic diseases is born at the end of pregnancies which have not abeen associated with an increased risk, where the risk of invasive procedures cannot be justified. Based on a method which we developed previously for sex-independent analysis of the fetal identity of cells in a fetal microchimeric setting, we plan to establish a technique allowing for non-invasive prenatal diagnosis of both numeric chromosomal aberrations and monogenetic hereditary diseases, using analysis of single cells. We will compare the efficiency of established protocols for enrichment of fetal cells from the peripheral blood of pregnant women. Furthermore, we will establish whole genome amplification of single cells and combine the analysis of fetal identity with analysis of trisomy by means of array comparative genomic hybridization. As a proof of principle for feasibility of analysis of monogenic hereditary diseases using the preamplified genome of single cells we will analyze mutations of the CFTR gene. The results of this work will be commercially exploited as we plan to establish an supra-regional center for cell- based non-invasive prenatal diagnosis by using the facilities of the Medical University of Graz.

The aim of the project was to evaluate the possibility for non-invasive prenatal diangosis on the basis of cells shed from the surface of the placenta (the trophoblast) into the maternal blood. In the course of a previous project we had tested an array of reagents (antibodies) allowing for detection of various types of trophoblast cells. Also, we had developed a technique for genotyping single cells, whether they contain the maternal of the fetal genome. During the course of the project we developed at first the method for analysis of single cells further: Amplification of the DNA was the basis for subsequent analysis combining genotyping with methods allowing for genetic diagnosis. Furthermore, we used a model system for comparing the efficiency of various methods for enrichment of trophoblast cells. We found that size based cell separation using a blood sieve with pores of a defined width was far superior to methods based on trophoblast-specific antibodies coupled to magnetic beads which allow for magnetic sorting of cells. Using size-based cell separation, the recovery of trophoblast-like cells spiked into blood in vitro attained