Detection of fetal trisomy and single gene disease by massively parallel sequencing of extracellular vesicle DNA in maternal plasma: a proof-of-concept validation

Abstract

BackgroundDuring human pregnancy, placental trophectoderm cells release extracellular vesicles (EVs) into maternal circulation. Trophoblasts also give rise to cell-free DNA (cfDNA) in maternal blood, and has been used for noninvasive prenatal screening for chromosomal aneuploidy. We intended to prove the existence of DNA in the EVs (evDNA) of maternal blood, and compared evDNA with plasma cfDNA in terms of genome distribution, fragment length, and the possibility of detecting genetic diseases.MethodsMaternal blood from 20 euploid pregnancies, 9 T21 pregnancies, 3 T18 pregnancies, 1 T13 pregnancy, and 2 pregnancies with FGFR3 mutations were obtained. EVs were separated from maternal plasma, and confirmed by transmission electronic microscopy (TEM), western blotting, and flow cytometry (FACS). evDNA was extracted and its fetal origin was confirmed by quantitative PCR (qPCR). Pair-end (PE) whole genome sequencing was performed to characterize evDNA, and the results were compared with that of cfDNA. The fetal risk of aneuploidy and monogenic diseases was analyzed using the evDNA sequencing data.ResultsEVs separated from maternal plasma were confirmed with morphology by TEM, and protein markers of CD9, CD63, CD81 as well as the placental specific protein placental alkaline phosphatase (PLAP) were confirmed by western blotting or flow cytometry. EvDNA could be successfully extracted for qPCR and sequencing from the plasma EVs. Sequencing data showed that evDNA span on all 23 pairs of chromosomes and mitochondria, sharing a similar distribution pattern and higher GC content comparing with cfDNA. EvDNA showed shorter fragments yet lower fetal fraction than cfDNA. EvDNA could be used to correctly determine fetal gender, trisomies, and de novo FGFR3 mutations.ConclusionsWe proved that fetal DNA could be detected in EVs separated from maternal plasma. EvDNA shared some similar features to plasma cfDNA, and could potentially be used to detect genetic diseases in fetus.

Clinical Significance of Non-Invasive Prenatal Screening for Trisomy 7: Cohort Study and Literature Review

“…In addition, there are very few studies about detecting rare autosomal trisomies using fetal cells present in maternal blood [ 7 ]. Recently, cell-free DNA (cfDNA) in maternal circulation, which is released by placenta trophoblast cells as well as DNA in the extracellular vesicles (evDNA) [ 8 , 9 ], is widely used for noninvasive prenatal screening (NIPS, or non-invasive prenatal testing) for common aneuploidies in the clinical setting.…”

Role of Extracellular Vesicles in Epithelial Ovarian Cancer: A Systematic Review

“…Extracellular vesicles (EVs) are a heterogeneous group of cell-derived submicron vesicles released under physiological or pathological conditions [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. In the past decade, EVs have been classified on the basis of their size and mechanisms of biogenesis, as exosomes (30–100 nm), when generated within endosomal compartments, microvesicles (MVs, 100–1000 nm), and apoptotic bodies (0.1–5 µm), both budding directly from the plasma membrane [ 18 ].…”

Characterizing Extracellular Vesicles and Their Diverse RNA Contents

“…Studying EVs may provide a distinct advantage over a total extracellular RNA approach since EV-contained RNAs can undergo RNA-unshielding and thereby carry a higher functional potential than when protein-bound in circulation (Nabet et al, 2017 ). Furthermore, EVs have demonstrated extensibility to sample from diverse cellular compartments including the nucleus and mitochondria (Yokoi et al, 2019 ; Zhang W. et al, 2019 ). Yokoi et al detected increased nuclear content (genomic DNA) loading into EVs within blood from ovarian cancer patients and in cell lines exposed to genotoxic drug treatment in vitro (Yokoi et al, 2019 ).…”

“…Deep and high-coverage sequencing studies of the fetal exome and genome have demonstrated the potential of extracellular RNA to make clinical assessments of the fetal genome at various time points (Fan et al, 2012 ). Recent studies affiliated with the sequencing company BGI Genomics have indicated that low-coverage (0.25×) sequencing of EV-derived DNA from only 250 μL of maternal blood plasma can cover all chromosomes, and notably, provided twice as much sampling of the mitochondrial genome than a cell-free approach (Zhang W. et al, 2019 ). Given the differential expression of genes within EVs vs. cell-free fluids, we believe that there is considerable potential for an EV-based approach for prenatal clinical assessment.…”