Noninvasive prenatal detection of fetal trisomy and single gene disease by shotgun sequencing of placenta originated exosome DNA: a proof-of-concept validation

Background: During human pregnancy, Placental trophectoderm cells can release exosomes into maternal circulation. Trophoblast cells also give rise to cell-free DNA (cfDNA) and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of exosomal DNA (exoDNA) in the exosomes of maternal blood and compared exoDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases. Methods: Maternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy and 2 pregnancies with FGFR3 mutations were obtained. Exosomes enriched from maternal plasma were confirmed by transmission electronic microscopy (TEM), western blotting and flow cytometry. ExoDNA was extracted and its fetal origin was confirmed by realtime fluorescence quantitative PCR(Q-PCR). Besides, exoDNA content was uncovered by Q-PCR. To characterize exoDNA and compare with cfDNA, pair-end whole genome sequencing was performed. Lastly, the fetal risk of genetic diseases was analyzed using the exoDNA sequencing data. Results: ExoDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. ExoDNA showed shorter fragments yet lower fetal fraction than cfDNA. ExoDNA could be used to determine fetal gender correctly, and all trisomies as well as de novo FGFR3 mutations. Conclusions: We proved that fetal exoDNA could be identified in the exosomes extracted from maternal plasma. ExoDNA shared some similar features to cfDNA and could potentially be used to detect genetic diseases in fetus.