Deborah G. Maddocks

Quantification of cell free fetal DNA in maternal plasma in normal pregnancies and in pregnancies with placental dysfunction.

OBJECTIVE To assess the normal levels of free fetal DNA in maternal plasma through pregnancy compared with those in pregnancies complicated with placental dysfunction manifested by preeclampsia and/or fetal growth restriction. STUDY DESIGN Maternal blood samples from 138 singleton male pregnancies were divided into 3 groups; normal pregnancies (77), preeclampsia (49), and fetal growth restriction (12). Royston and Wrights methods were used to calculate gestational age-related reference limits of free fetal DNA in the 3 groups. The DYS14 gene of the Y chromosome was quantified and compared in maternal plasma by using real-time quantitative polymerase chain reaction. RESULTS Free fetal DNA in normal pregnancies increased with gestational age. Results were significantly higher in preeclampsia and fetal growth restriction groups than in normal pregnancy and were higher in severe preeclampsia than in milder disease. CONCLUSION Free fetal DNA is a potential marker for placental dysfunction in pregnancy. Large prospective studies are now needed to investigate its role in the prediction of pregnancy complications and severity and or timing of delivery.

Molecular biology of Rh proteins and relevance to molecular medicine

The Rhesus (Rh) blood group system is expressed by a pair of 12-transmembrane-domain-containing proteins, the RhCcEe and RhD proteins. RhCcEe and RhD associate as a Rh core complex that comprises one RhD/CcEe protein and most likely two Rh-associated glycoproteins (RhAG) as a trimer. All these Rh proteins are homologous and share this homology with two human non-erythroid proteins, RhBG and RhCG. All Rh protein superfamily members share homology and function in a similar manner to the Mep/Amt ammonium transporters, which are highly conserved in bacteria, plants and invertebrates. Significant advances have been made in our understanding of the structure and function of Rh proteins, as well as in the clinical management of Rh haemolytic disease. This review summarises our current knowledge concerning the molecular biology of Rh proteins and their role in transfusion and pregnancy incompatibility.

The SAFE project: towards non-invasive prenatal diagnosis.

After the revolutionary detection of ffDNA (free fetal DNA) in maternal circulation by real-time PCR in 1997 and advances in molecular techniques, NIPD (non-invasive prenatal diagnosis) is now a clinical reality. Non-invasive diagnosis using ffDNA has been implemented, allowing the detection of paternally inherited alleles, sex-linked conditions and some single-gene disorders and is a viable indicator of predisposition to certain obstetric complications [e.g. PET (pre-eclampsia)]. To date, the major use of ffDNA genotyping in the clinic has been for the non-invasive detection of the pregnancies that are at risk of HDFN (haemolytic disease of the fetus and newborn). This has seen numerous clinical services arising across Europe and many large-scale NIPD genotyping studies taking place using maternal plasma. Because of the interest in performing NIPD and the speed at which the research in this area was developing, the SAFE (Special Non-Invasive Advances in Fetal and Neonatal Evaluation) NoE (Network of Excellence) was founded. The SAFE project was set up to implement routine, cost-effective NIPD and neonatal screening through the creation of long-term partnerships within and beyond the European Community and has played a major role in the standardization of non-invasive RHD genotyping. Other research using ffDNA has focused on the amount of ffDNA present in the maternal circulation, with a view to pre-empting various complications of pregnancy. One of the key areas of interest in the non-invasive arena is the prenatal detection of aneuploid pregnancies, particularly Downs syndrome. Owing to the high maternal DNA background, detection of ffDNA from maternal plasma is very difficult; consequently, research in this area is now more focused on ffRNA to produce new biomarkers.

Post-genomics studies and their application to non-invasive prenatal diagnosis.

Non-invasive prenatal diagnosis (NIPD) offers the opportunity to eliminate completely the risky procedures of amniocentesis and chorionic villus sampling. The development of NIPD tests has largely centred around the isolation and analysis of fetal cells in the maternal circulation and the analysis of free fetal DNA in maternal plasma. Both of these techniques offer difficult technical challenges, and at the current moment in time the use of free fetal DNA is the simplest and most effective method of defining paternally inherited fetal genes for diagnosis. Post-genomics technologies that explore the proteins (proteomics) and transcripts (transcriptomics) released by the placenta into the maternal circulation offer new opportunities to identify genes and their protein products that are key diagnostic markers of disease (in particular Down syndrome), and might replace the current screening markers in use for prediction of risk of Down syndrome. In the ideal situation, these markers are sufficiently diagnostic not to require invasive sampling of fetal genetic material. Post-genomics techniques might also offer better opportunities for defining fetal cell-specific markers that might enhance their isolation from maternal blood samples. This review describes progress in these studies, particularly those funded by the Special Non-invasive Advances in Fetal and Neonatal Evaluation (SAFE) Network of Excellence.

Cell-free fetal DNA in the maternal serum and plasma: current and evolving applications

Purpose of review Free fetal nucleic acids, found in the plasma of every pregnant woman, have made a substantial impact on prenatal diagnosis. The past decade has seen the introduction of routine noninvasive prenatal diagnosis (NIPD) using DNA extracted from maternal plasma for a number of clinical complications of pregnancy, notably feto-maternal blood group incompatibility, fetal sexing and exclusion/detection of single-gene disorders. It appears that mass-scale analysis of all RhD-negative pregnant women will be adopted to conserve stocks of prophylactic anti-D and avoid the administration of a blood product unnecessarily. For the majority of prenatal diagnostic procedures, the assessment of trisomy, particularly trisomy 21, is the highest priority. Because RHD genotyping, fetal sexing and analysis of single-gene disorders all depend on the detection of paternally inherited alleles, they were relatively simple to adapt on the basis of PCR analysis of DNA obtained from maternal plasma. However, for assessment of chromosome copy number, this is not so straightforward. Recent findings The assessment of polymorphisms among placentally expressed mRNAs found in maternal plasma has enabled the detection of trisomy 21 fetuses using a combination of reverse transcriptase PCR and mass spectrometry to define allelic ratios of maternally and paternally inherited single nucleotide polymorphisms. Interesting recent developments also include the finding that direct sequence analysis of maternal plasma extracted DNA using ‘next-generation’ DNA sequencers can differentiate between normal and trisomy fetuses. Summary NIPD using nucleic acids obtained from maternal plasma and serum is now a clinical reality, particularly in the management of hemolytic disease of the fetus and newborn. Recent advances signal that NIPD for common aneuploidies will soon be possible.

Quantification of free fetal DNA in multiple pregnancies and relationship with chorionicity

Free fetal DNA (ffDNA) in the maternal plasma appears to originate mainly from the trophoblast. We tested the hypothesis that ffDNA concentration is increased in multiple pregnancies where trophoblastic mass has been shown to be increased.

Quantification of circulatory fetal DNA in the plasma of pregnant women.

The analysis of cell-free fetal DNA in the circulation of the pregnant woman plays the pivotal role in noninvasive prenatal research. Here, we describe an improved method for the quantification of male DNA, which is a valuable research tool for the quantification of fetal DNA. The quantification of fetal DNA serves two main purposes. First, the levels may indicate certain pregnancy-related disorders such as preeclampsia even before onset of the disease; thus, the quantification may serve as a marker for early detection. Second, extraction and enrichment strategies of the fetal DNA compartment are important factors in the development and implementation of clinical tests, such as detection of fetal sex, Rhesus D status, point mutations, and aneuploidies. In this context, the quantification of fetal DNA is an important tool for the evaluation of protocols.