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

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Weiting Zhang ◽  
Sen Lu ◽  
Dandan Pu ◽  
Haiping Zhang ◽  
Lin Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blotting ◽  
Massively Parallel Sequencing ◽  
Genetic Diseases ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Similar Distribution ◽  
Placental Alkaline Phosphatase ◽  
Sequencing Data ◽  
Monogenic Diseases

Abstract Background During 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. Methods Maternal 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. Results EVs 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. Conclusions We 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.

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

2018 ◽  
Author(s):  
Weiting Zhang ◽  
Sen Lu ◽  
Jia Zhao ◽  
Dandan Pu ◽  
Haiping Zhang ◽  
...  
Keyword(s):  
De Novo ◽  
Single Gene ◽  
Genetic Diseases ◽  
Gc Content ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Similar Distribution ◽  
Sequencing Data ◽  
Maternal Circulation ◽  
Fgfr3 Mutations

BackgroundDuring 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.MethodsMaternal 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.ResultsExoDNA 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.ConclusionsWe 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.

scholarly journals Noninvasive Prenatal Diagnosis of Monogenic Diseases by Targeted Massively Parallel Sequencing of Maternal Plasma: Application to β-Thalassemia

2012 ◽  
Vol 58 (10) ◽  
pp. 1467-1475 ◽  
Author(s):  
Kwan-Wood G Lam ◽  
Peiyong Jiang ◽  
Gary J W Liao ◽  
K C Allen Chan ◽  
Tak Y Leung ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Massively Parallel Sequencing ◽  
Globin Gene ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Sequencing Data ◽  
Plasma Dna ◽  
Parallel Sequencing ◽  
Noninvasive Prenatal Diagnosis ◽  
Monogenic Diseases

Abstract BACKGROUND A genomewide genetic and mutational profile of a fetus was recently determined via deep sequencing of maternal plasma DNA. This technology could have important applications for noninvasive prenatal diagnosis (NIPD) of many monogenic diseases. Relative haplotype dosage (RHDO) analysis, a core step of this procedure, would allow one to elucidate the maternally inherited half of the fetal genome. For clinical applications, the cost and complexity of data analysis might be reduced via targeted application of this approach to selected genomic regions containing disease-causing genes. There is thus a need to explore the feasibility of performing RHDO analysis in a targeted manner. METHODS We performed target enrichment by using solution-phase hybridization followed by massively parallel sequencing of the β-globin gene region in 2 families undergoing prenatal diagnosis for β-thalassemia. We used digital PCR strategies to physically deduce parental haplotypes. Finally, we performed RHDO analysis with target-enriched sequencing data and parental haplotypes to reveal the β-thalassemic status for the fetuses. RESULTS A mean sequencing depth of 206-fold was achieved in the β-globin gene region by targeted sequencing of maternal plasma DNA. RHDO analysis was successful for the sequencing data obtained from the target-enriched samples, including a region in one of the families in which the parents had similar haplotype structures. Data analysis revealed that both fetuses were heterozygous carriers of β-thalassemia. CONCLUSIONS Targeted sequencing of maternal plasma DNA for NIPD of monogenic diseases is feasible.

Non-invasive prenatal testing for fetal aneuploidy by massively parallel DNA sequencing of maternal plasma: the future has arrived today/Nicht-invasiver Pränatal-Test auf fetale Aneuploidie mittels massiv-paralleler DNA-Sequenzanalyse im mütterlichen Plasma: in der Zukunft angekommen

2012 ◽  
Vol 36 (5) ◽  
Author(s):  
Amy Swanson ◽  
Christin Coffeen ◽  
Amy J. Sehnert
Keyword(s):  
Massively Parallel Sequencing ◽  
Prenatal Testing ◽  
The United States ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Massively Parallel ◽  
Clinical Implementation ◽  
Maternal Cell ◽  
Non Invasive ◽  
Fetal Aneuploidy

AbstractAfter decades of research, non-invasive prenatal testing (NIPT) using maternal blood to determine fetal chromosome status has found its way from the research laboratory into clinical practice, triggering a long-awaited paradigm shift in prenatal care. A variety of methods using sequencing of maternal cell-free DNA (cfDNA) have now been studied, primarily demonstrating their ability to detect the most common fetal aneuploidy, trisomy 21 (T21). The focus of this article is on massively parallel sequencing (MPS) with optimized sequence tag mapping and chromosome quantification, which accurately detects T21 as well as multiple other aneuploidies across the genome. The power of this technique resides in its high precision and reduction of variation within and between sequencing runs. Using MPS, classification of aneuploidy status for a given sample can be reliably assigned from the genetic information alone without the need to factor in other maternal pre-test risk or other clinical variables. Performance of this method has been prospectively demonstrated in a rigorous, blinded, multi-center study in the United States. The findings suggest that MPS can be incorporated into existing prenatal screening algorithms to reduce unnecessary invasive procedures. This technology and key considerations for clinical implementation are discussed.

Effect of preexamination conditions in a centralized-testing model of non-invasive prenatal screening

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Chad Fibke ◽  
Sylvie Giroux ◽  
André Caron ◽  
Elizabeth Starks ◽  
Jeremy D.K. Parker ◽  
...  
Keyword(s):  
Gestational Age ◽  
Prenatal Testing ◽  
Positive Association ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Maternal Body Mass Index ◽  
Sequencing Data ◽  
Non Invasive ◽  
Tube Type ◽  
Sequencing Platforms

Abstract Objectives Non-invasive prenatal testing requires the presence of fetal DNA in maternal plasma. Understanding how preexamination conditions affect the integrity of cell-free DNA (cfDNA) and fetal fraction (FF) are a prerequisite for test implementation. Therefore, we examined the adjusted effect that EDTA and Streck tubes have on the cfDNA quantity and FF. Methods A total of 3,568 maternal blood samples across Canada were collected in either EDTA, or Streck tubes, and processing metrics, maternal body mass index (BMI), gestational age and fetal karyotype and sex were recorded. Plasma samples were sequenced using two different sequencing platforms in separate laboratories. Sequencing data were processed with SeqFF to estimate FF. Linear regression and multivariate imputation by chained equations were used to estimate the adjusted effect of tube type on cfDNA and FF. Results We found a positive association between cfDNA quantity and blood shipment time in EDTA tubes, which is significantly reduced with the use of Streck tubes. Furthermore, we show the storage of plasma at −80 °C is associated with a 4.4% annual relative decrease in cfDNA levels. FF was not associated with collection tube type when controlling for confounding variables. However, FF was positively associated with gestational age and trisomy 21, while negatively associated with BMI, male fetus, trisomy 18, Turners syndrome and triploidy. Conclusions Preexamination, maternal and fetal variables are associated with cfDNA quantity and FF. The consideration of these variables in future studies may help to reduce the number of pregnant women with inconclusive tests as a result of low FF.

scholarly journals Noninvasive Prenatal Methylomic Analysis by Genomewide Bisulfite Sequencing of Maternal Plasma DNA

2013 ◽  
Vol 59 (11) ◽  
pp. 1583-1594 ◽  
Author(s):  
Fiona MF Lun ◽  
Rossa WK Chiu ◽  
Kun Sun ◽  
Tak Y Leung ◽  
Peiyong Jiang ◽  
...  
Keyword(s):  
Bisulfite Sequencing ◽  
Biomarker Discovery ◽  
Prenatal Development ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Methylation Profile ◽  
Sequencing Data ◽  
Plasma Dna ◽  
Blood Samples ◽  
Fetal Dna

BACKGROUND Epigenetic mechanisms play an important role in prenatal development, but fetal tissues are not readily accessible. Fetal DNA molecules are present in maternal plasma and can be analyzed noninvasively. METHODS We applied genomewide bisulfite sequencing via 2 approaches to analyze the methylation profile of maternal plasma DNA at single-nucleotide resolution. The first approach used maternal blood samples and polymorphic differences between the mother and fetus to analyze the fetal methylome across the genome. The second approach used the methylation profile of maternal blood cells and the fractional fetal DNA concentration in maternal plasma to deduce the placental methylomic profile from maternal plasma DNA-sequencing data. RESULTS Because of the noninvasive nature of these approaches, we were able to serially assess the methylation profiles of fetal, placental, and maternal plasma with maternal blood samples collected in the first and third trimesters and after delivery. Gestation-related changes were observed. The fetal methylation profile deduced from maternal plasma data resembled that of the placental methylome, both on a genomewide level and per CpG site. Imprinted genes and differentially methylated regions were identified from the maternal plasma data. We demonstrated one potential clinical application of maternal plasma bisulfite sequencing with the successful detection of fetal trisomy 21. CONCLUSIONS We successfully analyzed fetal and placental methylomes on a genomewide scale, noninvasively and serially. This development offers a powerful method for research, biomarker discovery, and clinical testing for pregnancy-related disorders.

Non-invasive prenatal diagnosis of monogenic disorders: an optimized protocol using MEMO qPCR with miniSTR as internal control

2015 ◽  
Vol 53 (2) ◽  
Author(s):  
Claire Guissart ◽  
Vanessa Debant ◽  
Marie Desgeorges ◽  
Corinne Bareil ◽  
Caroline Raynal ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Internal Control ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Developmental Model ◽  
Dna Profile ◽  
Monogenic Disorders ◽  
Non Invasive ◽  
Cell Free Fetal Dna ◽  
Monogenic Diseases

AbstractAnalysis of circulating cell-free fetal DNA (cffDNA) in maternal plasma is very promising for early diagnosis of monogenic diseases. However, this approach is not yet available for routine use and remains technically challenging because of the low concentration of cffDNA, which is swamped by the overwhelming maternal DNA.To make clinical applications more readily accessible, we propose a new approach based on mutant enrichment with 3′-modified oligonucleotides (MEMO) PCR along with real-time PCR to selectively amplify from the maternal blood the paternally inherited fetal allele that is not present in the maternal genome.The first proof of concept of this strategy was displayed for cystic fibrosis by the accuracy of our detection of the p.Gly542* mutation used as the initial developmental model. Subsequently, a retrospective study of plasmas originating from two pregnant women carrying a fetus with private mutation confirmed the effectiveness of our method. We confirmed the presence of cffDNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit.This new non-invasive prenatal diagnosis test offers numerous advantages over current methods: it is simple, cost effective, time efficient and does not require complex equipment or bioinformatics settings. Moreover, our assays for different private mutations demonstrate the viability of this approach in clinical settings for monogenic disorders.

scholarly journals Creating basis for introducing NIPT in the Estonian public health setting

2018 ◽  
Author(s):  
Olga Žilina ◽  
Kadri Rekker ◽  
Lauris Kaplinski ◽  
Martin Sauk ◽  
Priit Paluoja ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Clinical Care ◽  
Maternal Blood ◽  
Whole Genome ◽  
Sequencing Data ◽  
Analysis Pipeline ◽  
Genome Library ◽  
Two Samples ◽  
Fetal Aneuploidies ◽  
Public Health Setting

AbstractObjectiveThe study aimed to validate a whole-genome sequencing-based NIPT method and our newly developed NIPTmer analysis software with the potential to integrate the pipeline into prenatal clinical care in Estonia.MethodIn total, 447 maternal blood samples were included to the study. Analysis pipeline involved whole-genome library preparation and massively parallel sequencing on Illumina NextSeq 500. Aneuploidy status was determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data. To estimate fetal fraction (FF) from total cell-free DNA SeqFF was implemented.ResultsNIPTmer software allowed to identify correctly all samples of non-mosaic T21 (15/15), T18 (9/9) and T13 (4/4) cases. However, one mosaic T18 remained undetected. Six false positive results were observed, including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). FF < 4% (2.8-3.99%) was estimated in eight samples, including two samples with T13 and T18. Despite low FF, these two samples were determined as aneuploid with NIPTmer software.ConclusionOur NIPT analysis pipeline proved to perform efficiently in detecting common fetal aneuploidies T21, T18 and T13 and is feasible for implementation into clinical service in Estonia.

scholarly journals Circulating Fetal Cell-Free DNA Fractions Differ in Autosomal Aneuploidies and Monosomy X

2014 ◽  
Vol 60 (1) ◽  
pp. 243-250 ◽  
Author(s):  
Richard P Rava ◽  
Anupama Srinivasan ◽  
Amy J Sehnert ◽  
Diana W Bianchi
Keyword(s):  
Massively Parallel Sequencing ◽  
Prenatal Testing ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Maternal Body Mass Index ◽  
Cell Free Dna ◽  
Monosomy X ◽  
Free Dna ◽  
Human Genome Assembly ◽  
Diagnostic Sensitivity And Specificity

Abstract BACKGROUND Noninvasive prenatal testing based on massively parallel sequencing (MPS) of cell-free DNA in maternal plasma has become rapidly integrated into clinical practice for detecting fetal chromosomal aneuploidy. We directly determined the fetal fraction (FF) from results obtained with MPS tag counting and examined the relationships of FF to such biological parameters as fetal karyotype and maternal demographics. METHODS FF was determined from samples previously collected for the MELISSA (Maternal Blood Is Source to Accurately Diagnose Fetal Aneuploidy) study. Samples were resequenced, analyzed blindly, and aligned to the human genome (assembly hg19). FF was calculated in pregnancies with male or aneuploid fetuses by means of an equation that incorporated the ratio of the tags in these samples to those of a euploid training set. RESULTS The mean (SD) FF from euploid male pregnancies was 0.126 (0.052) (n = 160). Weak but statistically significant correlations were found between FF and the maternal body mass index (r2 = 0.18; P = 2.3 × 10−8) and between FF and gestational age (r2 = 0.02; P = 0.047). No relationship with maternal ethnicity or age was observed. Mean FF values for trisomies 21 (n = 90), 18 (n = 38), and 13 (n = 16) and for monosomy X (n = 20) were 0.135 (0.051), 0.089 (0.039), 0.090 (0.062), and 0.106 (0.045), respectively. CONCLUSIONS MPS tag-count data can be used to determine FF directly and accurately. Compared with male euploid fetuses, the FF is higher in maternal plasma when the fetus has trisomy 21 and is lower when the fetus has trisomy 18, 13, or monosomy X. The different biologies of these aneuploidies have practical implications for the determination of cutoff values, which in turn will affect the diagnostic sensitivity and specificity of the test.

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