Cell-free Nucleic Acids in Maternal Plasma

The invasive procedures amniocentesis and chorionic villus sampling are routinely applied in pregnancies at risk for fetal genetic disorders and the results obtained are the gold standard for prenatal diagnosis. These procedures have an approximately 0.5-1% risk for fetal loss and are mainly used in cases at risk for fetal chromosomal abnormalities and single-gene disorders. Identification of cell-free fetal nucleic acids (DNA and RNA) in maternal plasma and the recognition that they represent a useful source of fetal genetic material for prenatal diagnosis has led to intensive efforts to develop non-invasive prenatal testing. This review summarizes recent developments in the field of non-invasive prenatal diagnosis through the use of cell-free fetal nucleic acids in maternal circulation during pregnancy and provides an overview of the possibilities for future clinical applications. Cell-free Nucleic Acids in Maternal Plasma The year 1997 is a landmark for non-invasive prenatal diagnosis (PD) as it was hailed the identification of the presence of cell-free fetal DNA (cffDNA) in the maternal circulation that opened new horizons for non-invasive PD (1). cffDNA originates from the placenta as a result of trophoblastic apoptosis. A less likely source, due to its scarcity, is fetal cells that undergo apoptosis in the maternal circulation. It can be found in low concentrations in the maternal plasma and cffDNA molecules are outnumbered 20:1 by maternal cell-free DNA molecules. cffDNA constitutes approximately 3-6% of the total cell-free DNA present in the maternal circulation (2). Recent data, however, show that its actual proportion is approximately 19% (3). It can be detected in maternal plasma from the 6th week of gestation, with increasing concentration throughout gestation. A large number of studies showed that postpartum, cffDNA is rapidly cleared from the maternal circulation with a half-life of approximately 16.3 min (4). Moreover, it has a relatively smaller size compared to maternal cell-free DNA, offering an alternative enrichment option (5). Its size varies between individuals and because half of the fetal genome is of maternal origin, its use in the diagnosis of fetal aneuploidies and single gene disorders is problematic. Three years after the discovery of cffDNA Poon et al. isolated cffRNA from maternal plasma using a reversetranscriptase polymerase chain reaction (PCR) (6). In contrast to the instability of RNA molecules, cffRNA is relatively stable because of its storage within apoptotic microvesicles in the plasma, which protect it from RNase degradation. It appears in the maternal circulation as early as the 4th week of gestation and rapidly disappears from maternal plasma after delivery, having a median half-life of 14 minutes. The main advantage of cffRNA over cffDNA is that it is possible to select for placenta-specific mRNA sequences not expressed by any maternal tissues (7). Approaches to cffDNA and -RNA Analysis Both maternal plasma and serum contain cffDNA, plasma however is the material of choice for PD since it contains less maternal background. Plasma DNA isolation is performed manually with commercially available kits, although automation of the process has also been reported (8). The scarcity of cffDNA in maternal plasma and its coexistence with maternal DNA represent the two major limitations for the use of cffDNA for diagnosis. Various methods have been used to overcome these limitations, including those based on the fact that fetal fragments tend to be of shorter length than maternal fragments (5). Efforts to increase the relative proportion of fetal DNA compared to 165 Correspondence to: Aristeidis G. Vaiopoulos, MD, M.Sc., Department of Medical Genetics, Athens University School of Medicine, Aghia Sofia Children’s Hospital, Thivon & Levadias, 115 27, Athens, Greece. Tel: +3