Comparing efficiency of cell free DNA and exosomal DNA as non-invasive screening method in prenatal diagnosis
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naeimeh shibaei,1,*
1. Islamic Azad university Shabestar branch
- Introduction: Prenatal genetic diagnosis relies on invasive methods to obtain fetal material suitable for analysis, such as amniocentesis and chorionic villi sampling. These procedures carry a small but significant risk of fetal loss, as well as risk to the mother alone (around 1% – 2% of cases). The discovery of cell free fetal DNA (cffDNA) and exosomal DNA (exoDNA) in the blood of pregnant women leads to immediate recognition as an alternative source of fetal material for prenatal diagnosis. It also leads to the formation of a novel field of non-invasive prenatal diagnosis (NIPD).
- Methods: In this article, we aim to summarize the fast-growing evidence for cffDNA and exosomal DNA as minimally invasive screening method in prenatal diagnosis and to highlight their opposing diagnostic advantages and disadvantages.
- Results: Placenta trophoblast cells are known to release cell-free DNA (cfDNA) into maternal circulation, which are DNA fragments of 160-170 base pair. The concentration of placental cfDNA in plasma, also known as the fetal fraction, is on average 10% of the total plasma cfDNA, and increases with gestational weeks. Trophoblast cells also release exosomes into maternal circulation. Exosomes are a type of extracellular vesicles (EV) that contain constituents (protein, DNA, and RNA) of the cells that secrete them. In 2018, the existence of exosomal DNA (exoDNA) in the exosomes of maternal blood was proven. They could identify fetal exoDNA in the exosomes extracted from maternal plasma. ExoDNA show shorter fragments yet lower fetal fraction than cfDNA. The median fragment size of exoDNA is 152.4bp with the standard deviation of ±10.51bp and the exosome concentration positively correlates with gestation weeks.
cell-free DNA (cfDNA) became a sensitive biomarker for detection of fetal DNA (cffDNA) in noninvasive prenatal test. ExoDNA shared some similar features to plasma cffDNA and could potentially be used to noninvasively detect fetal conditions such as chromosome aneuploidy and single gene disease. It has been proven that, exoDNA could be used to determine fetal gender correctly, and all trisomies as well as mutations that cause genetic diseases in fetus.
CfDNA is known to be a fragmented DNA and prone to nuclease activity in the circulation. The estimated half-life of cfDNA in the peripheral blood ranges between 4 min and 12 h. this short half life forcing fast protocols of sample collection. On the contrary, EVs and their cargo show a considerable long-term stability in body fluids that facilitate their analysis in biobanked samples.
Maternal plasma contain not only maternal but also fetal exoDNA. Although the fetal fraction of exoDNA have a nice correlation to that of cfDNA, it is significantly lower than cfDNA and with weak relationship to gestational weeks. Theoretically, using the placenta specific markers such as PLAP, pure placenta-derived exosomes can be specifically isolate. This may raise huge benefits to develop novel NIPT methods to detect copy number variation and monogenetic disease in fetus, since the high fetal fraction of placental exoDNA can overcome the current NIPT limitations that are mainly caused by the mixture of maternal-fetal cfDNA and low fetal fraction.
CfDNA has proven remarkable performance in next-generation sequencing techniques (NSG) and studies based in gene-wide analysis (GWA). Nowadays, the use of PCR-based assays that can simultaneously assess multiple regions of driver genes and droplet digital PCR enables screening tests of fetal deasease.
- Conclusion: Although, both exoDNA and cfDNA could be applied to the noninvasive prenatal studies with a real-time PCR method and other DNA quentity assessing methods and the use of EV-DNA did not lead to improved sensitivity or better detection of prenatal disease, considering the known difficulties during isolation and stability of cfDNA, exosomes might provide a new opportunity for prenatal diagnosis and screening.
- Keywords: cell free fetal DNA (cffDNA)
exosomal DNA (exoDNA)
non-invasive prenatal diagnosis (NIPD)
