Genetic analysis of fetal cells in maternal blood

This thesis investigates the development of noninvasive prenatal diagnosis using fetal cells in maternal circulation based on the polymerase chain reaction (PCR). The first section of the thesis discusses the theoretical issues of PCR-based noninvasive prenatal diagnosis.

The demonstration of the feasibility of this approach was through the detection of Y-chromosomal sequences from male fetuses by nested PCR amplification from maternal blood DNA. Fetal DNA was found to be detectable throughout the three trimesters of pregnancy. These observations open up the field of noninvasive prenatal diagnosis of single gene disorders. Further investigations showed that most women have cleared their circulation of fetal cells to below 1 fetal cell in 300,000 maternal nucleated cells by 8 weeks post-partum. The possibility of improving the accuracy of the method by multiple analyses was also explored.

The further development and streamlining of the assay was carried out by incorporating the process of Hot Start PCR, dUTP-based amplification and uracil-N-glycosylase (UNG) treatment and the use of new amplification primers. The value of the PCR in the development of fetal cell enrichment strategies was also illustrated.

The use of this PR-based approach in the detection of autosomal fetal genes was then pursued by applying the method to the prenatal determination of fetal rhesus D status by analysis of peripheral blood of rhesus D negative mothers. The success of this approach suggests the possibility that this may be applied to the management of rhesus D sensitised pregnancies.

The next section of the thesis explores the difficult situation whereby the target for prenatal diagnosis is shared between the mother and the fetus; but with the fetal target differing by a small number of nucleotides from the maternal one. This issue was addressed by the development of restriction enzyme enhanced Amplification Refractory Mutation System (ARMS) and double ARMS, based on the prenatal diagnosis of cystic fibrosis and <IMG WIDTH=9 HEIGHT=24 ALIGN=MIDDLE SRC="/maths/beta.gif">-thalassaemia as model systems. The further application of double ARMS to the detection of post bone marrow transplantation chimaerism was also explored.(D180008)