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Genotype result interpretation. In example 1 and 2, the presence of the paternal-specific alleles 10 and 14 allows concluding that these two single cells are fetal cells. Example 3 illustrates the identification of a maternal cell on the basis of its genotype. EXP: expansion of triplet repeats; MAT: maternal cell; N: normal triplet repeat size; NIPD: non-invasive prenatal diagnosis; SFC: Single Fetal Cell.
Source publication
Non-Invasive Prenatal Diagnosis (NIPD), based on the analysis of circulating cell-free fetal DNA (cff-DNA), is successfully implemented for an increasing number of monogenic diseases. However, technical issues related to cff-DNA characteristics remain, and not all mutations can be screened with this method, particularly triplet expansion mutations...
Citations
... It provides a more accurate and comprehensive picture of what happens to an organism at a specific point in time, which will help us better understand diseases. Single-cell analysis has involved in many fields, including basic research applications, such as cancer [3], immunology [4], neurology [5], stem cells [6], etc., and in clinical applications such as non-invasive prenatal diagnosis [7], in vitro fertilization [8], and circulating tumor cells (CTCs) [9]. neurology [5], stem cells [6], etc., and in clinical applications such as non-invasive prenatal diagnosis [7], in vitro fertilization [8], and circulating tumor cells (CTCs) [9]. ...
... Single-cell analysis has involved in many fields, including basic research applications, such as cancer [3], immunology [4], neurology [5], stem cells [6], etc., and in clinical applications such as non-invasive prenatal diagnosis [7], in vitro fertilization [8], and circulating tumor cells (CTCs) [9]. neurology [5], stem cells [6], etc., and in clinical applications such as non-invasive prenatal diagnosis [7], in vitro fertilization [8], and circulating tumor cells (CTCs) [9]. ...
Single-cell analysis provides an overwhelming strategy for revealing cellular heterogeneity and new perspectives for understanding the biological function and disease mechanism. Moreover, it promotes the basic and clinical research in many fields at a single-cell resolution. A digital polymerase chain reaction (dPCR) is an absolute quantitative analysis technology with high sensitivity and precision for DNA/RNA or protein. With the development of microfluidic technology, digital PCR has been used to achieve absolute quantification of single-cell gene expression and single-cell proteins. For single-cell specific-gene or -protein detection, digital PCR has shown great advantages. So, this review will introduce the significance and process of single-cell analysis, including single-cell isolation, single-cell lysis, and single-cell detection methods, mainly focusing on the microfluidic single-cell digital PCR technology and its biological application at a single-cell level. The challenges and opportunities for the development of single-cell digital PCR are also discussed.
... These populations of cells can theoretically be isolated from peripheral maternal blood from as early as 6 weeks of pregnancy, although reportedly more reliably from 10-14 weeks, and offer a source of intact fetal chromosomal DNA for NIPD of monogenic conditions [140][141][142][143] . Obtaining a pure fetal cell population provides the benefit of having intact gDNA within which more complex pathogenic variants can be detected [144] , such as triplet repeat expansions [145] . Furthermore, obtaining high molecular weight fetal gDNA negates the need for RHDO or RMD as maternal background DNA is not present in this sample type. ...
Prenatal testing is important for the early detection and diagnosis of rare genetic conditions with life-changing implications for the patient and their family. Gaining access to the fetal genotype can be achieved using gold-standard invasive sampling methods, such as amniocentesis and chorionic villus sampling, but these carry a small risk of miscarriage. Non-invasive prenatal diagnosis (NIPD) for select rare monogenic conditions has been in clinical service in England since 2012 and has revolutionised the field of prenatal diagnostics by reducing the number of women undergoing invasive sampling procedures. Fetal-derived genomic material is present in a highly fragmented form amongst the maternal cell-free DNA (cfDNA) in circulation, with sequence coverage across the entire fetal genome. Cell-free fetal DNA (cffDNA) is the foundation for NIPD, and several technologies have been clinically implemented for the detection of paternally inherited and de novo pathogenic variants. Conversely, a low abundance of cffDNA within a high background of maternal cfDNA makes assigning maternally inherited variants to the fetal fraction a significantly more challenging task. Research is ongoing to expand available tests for maternal inheritance to include a broader range of monogenic conditions, as well as to uncover novel diagnostic avenues. This review covers the scope of technologies currently clinically available for NIPD of monogenic conditions and those still in the research pipeline towards implementation in the future.
... Trophoblast cells can be isolated non-invasively as early as 5 weeks of gestation, [58] and this cell type has recently gained popularity as one of the two cells of choice for cbNIPD. [59][60][61][62][63][64][65][66] But the yield of these cells from maternal blood is poor, and in one study, only half the cells isolated were of high quality and suitable for next-generation sequencing (NGS), whereas the rest were of poor quality, in S-phase or apoptotic. [67] Apoptotic trophoblast cells show loss of whole or segments of chromosomes, and produce noisy NGS signals, making clinical diagnosis difficult. ...
The options for prenatal genetic testing have evolved rapidly in the past decade, and advances in sequencing technology now allow genetic diagnoses to be made down to the single-base-pair level, even before the birth of the child. This offers women the opportunity to obtain information regarding the foetus, thereby empowering them to make informed decisions about their pregnancy. As genetic testing becomes increasingly available to women, clinician knowledge and awareness of the options available to women is of great importance. Additionally, comprehensive pretest and posttest genetic counselling about the advantages, pitfalls and limitations of genetic testing should be provided to all women. This review article aims to cover the range of genetic tests currently available in prenatal screening and diagnosis, their current applications and limitations in clinical practice as well as what the future holds for prenatal genetics.
... The immunoaffinity approach is the most common microfluidic method to isolate CFCs for NIPD. Nearly half of these studies targeted fnRBCs for NIPD [19,22,23], and the other half targeted EVTs [20,[24][25][26]. Only one study performed analysis of both fnRBCs and fEVT [21]. ...
... Caryrefourcq et al., used the DEPArray method to isolate fEVTs and used Huntington disease as a clinical model for cell-based NIPD in 2020. Among the seven participants, only five fEVTs were isolated from four pregnant women and only one got a conclusive NIPD [25]. ...
The purpose of the present review is to try to highlight recent advances in the application of microfluidic technology on non-invasive prenatal diagnosis (NIPD). The immunoaffinity based microfluidic technology is the most common approach for NIPD, followed by size-based microfluidic methods. Immunoaffinity microfluidic methods can enrich and isolate circulating fetal extravillous trophoblasts (fEVTs) or fetal nucleated red blood cells (fnRBCs) for NIPD by using specific antibodies, but size-based microfluidic systems are only applied to isolate fEVTs. Most studies based on the immunoaffinity microfluidic system gave good results. Enough fetal cells were obtained for chromosomal and/or genetic analysis in all blood samples. However, the results from studies using size-based microfluidic systems for NIPD are less than ideal. In conclusion, recent advances in microfluidic devices make the immunoaffinity based microfluidic system potentially a powerful tool for cell-based NIPD. However, more clinical validation is needed.
... 13 Due to their presence in maternal circulation during the first trimester of pregnancy, they became an attractive target for noninvasive prenatal testing (NIPT). 14,15 Previous investigations of methods for isolation of cEVTs resulted in laborious protocols with inconsistent and insufficient cell recovery 16 as the identification of cEVTs by manual needle-based cell picking or laser capture microdissection under the microscope is highly operator-dependent. 15,17,18 Recently, advances have been made in the single-cell sorting of cEVTs using flow cytometry, followed by Short-Tandem Repeats (STR) analysis. ...
... | DISCUSSIONThe semi-automated methodology presented here for the isolation and single-cell analysis of cEVTS from maternal blood supports the feasibility and scalability of a cell-based noninvasive prenatal test for fetal genomic profiling down to~1Mb in size. The goal of our work was to develop a new method that, different from others reported,[16][17][18][19] would(i) reduce hands-on manipulation of isolated fetal cells and (ii) both demonstrate the fetal origin by genetic confirmation (not only by immunophenotype) and perform fetal genome profiling for aneuploidy and subchromosomal variant in each isolated single cell. Current work is in progress to both validate our current workflow on a large patient cohort (over 1500 patients) to a single-cell analytical LoD of~1Mb and to increase the overall number of samples with recovery and the number of cEVTs isolated per sample. ...
Objective:
To develop a multi-step workflow for the isolation of cEVTs by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and CNV detection.
Methods:
Determination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with gDNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analysed to evaluate the current workflow.
Results:
A LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by CMA. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell.
Conclusion:
Our semi-automated methodology for the isolation and single cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling. This article is protected by copyright. All rights reserved.
... There are a number of reports in the literature reporting routine use for pregnancies where there is a relevant family history of a specific disorder [24][25][26][27]. There is ongoing translational research into circulating fetal cells and circulating fetal trophoblast cells (CFTCs) for NIPD of monogenic disease [28,29]. CFTCs could potentially be used for diagnosis of triplet repeat expansions or point mutations, variants which cffDNA cannot pick up. ...
Congenital malformations diagnosed by ultrasound screening complicate 3–5% of pregnancies and many of these have an underlying genetic cause. Approximately 40% of prenatally diagnosed fetal malformations are associated with aneuploidy or copy number variants, detected by conventional karyotyping, QF-PCR and microarray techniques, however monogenic disorders are not diagnosed by these tests. Next generation sequencing as a secondary prenatal genetic test offers additional diagnostic yield for congenital abnormalities deemed to be potentially associated with an underlying genetic aetiology, as demonstrated by two large cohorts: the ‘Prenatal assessment of genomes and exomes’ (PAGE) study and ‘Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study’ performed at Columbia University in the US. These were large and prospective studies but relatively ‘unselected’ congenital malformations, with little Clinical Genetics input to the pre-test selection process. This review focuses on the incremental yield of next generation sequencing in single system congenital malformations, using evidence from the PAGE, Columbia and subsequent cohorts, with particularly high yields in those fetuses with cardiac and neurological anomalies, large nuchal translucency and non-immune fetal hydrops (of unknown aetiology). The total additional yield gained by exome sequencing in congenital heart disease was 12.7%, for neurological malformations 13.8%, 13.1% in increased nuchal translucency and 29% in non-immune fetal hydrops. This demonstrates significant incremental yield with exome sequencing in single-system anomalies and supports next generation sequencing as a secondary genetic test in routine clinical care of fetuses with congenital abnormalities.
... Recently, automated fetal nRBC and extravillous trophoblast capture systems have been validated in the genetic diagnosis [45,46]. Nevertheless, insufficient clinical trials able to provide evidence demonstrating a robustness of cbDNA and its diagnostic value in fetal aneuploidy diagnosis still restrain its clinical implementation [47]. ...
Trisomy 21 (T21) is one of the most commonly occurring genetic disorders, caused by the partial or complete triplication of chromosome 21. Despite the significant progress in the diagnostic tools applied for prenatal screening, commonly used methods are still imprecise and involve invasive diagnostic procedures that are related to a maternal risk of miscarriage. In this case, novel prenatal biomarkers are still being evaluated using highly specialized techniques, which could increase the diagnostic usefulness of biochemical prenatal screening for T21. From the other hand, the T21′s pathogenesis, caused by the improper division of genetic material, disrupting many metabolic pathways, could be further evaluated with the use of omics methods, which could result in bringing relevant insights for the evaluation of potential medical targets. Accordingly, a literature search was undertaken to collect novel information about prenatal screening for Down syndrome with the use of advanced technology, with a particular emphasis on the evaluation of novel screening biomarkers and the discovery of potential medical targets. These meta-analyses are focused on novel approaches designed with the use of omics techniques, representing the most rapidly developing and promising field in research today. Considering the limitations and progress of these methods, the use of omics techniques in evaluating T21 pathogenesis could bring beneficial results in prenatal screening, simultaneously uncovering novel potential medical targets.
... To date, cbNIPT has been used to test for the carrier status of monogenic disorders [17,25,26] and for aneuploidy, unbalanced structural translocations and smaller deletions [23,27]. To our knowledge, only one previous publication has reported the use of cbNIPT following PGT-M, presenting data from two couples at risk of transmitting congenital deafness and ichthyosis caused by point mutations or smaller deletions [28]. ...
Purpose
Proof of concept of the use of cell-based non-invasive prenatal testing (cbNIPT) as an alternative to chorionic villus sampling (CVS) following preimplantation genetic testing for monogenic disorders (PGT-M).
Method
PGT-M was performed by combined testing of short tandem repeat (STR) markers and direct mutation detection, followed by transfer of an unaffected embryo. Patients who opted for follow-up of PGT-M by CVS had blood sampled, from which potential fetal extravillous throphoblast cells were isolated. The cell origin and mutational status were determined by combined testing of STR markers and direct mutation detection using the same setup as during PGT. The cbNIPT results with respect to the mutational status were compared to those of genetic testing of the CVS.
Results
Eight patients had blood collected between gestational weeks 10 and 13, from which 33 potential fetal cell samples were isolated. Twenty-seven out of 33 isolated cell samples were successfully tested (82%), of which 24 were of fetal origin (89%). This corresponds to a median of 2.5 successfully tested fetal cell samples per case (range 1–6). All fetal cell samples had a genetic profile identical to that of the transferred embryo confirming a pregnancy with an unaffected fetus, in accordance with the CVS results.
Conclusion
These findings show that although measures are needed to enhance the test success rate and the number of cells identified, cbNIPT is a promising alternative to CVS.
Trial registration number
N-20180001
... This is because cell-based NIPT is still feasible even lacking a portion of genomic DNA of a cell by merging sequencing data from more than one cell. When 50% of the entire genome is covered without allelic dropout by the sequencing data from one cell on average, 23 Therefore, replacement of EDTA tubes with Streck tubes for maternal blood sampling is expected to protect fNRBCs from their loss immediately after blood sampling and to improve DNA amplification efficiencies at latter procedures. ...
Purpose
Although non-invasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false-negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT.
Methods
Using male-bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y-chromosome marker and whole-genome amplification (WGA)-based whole-genome sequencing.
Results
The PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA-based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA.
Conclusions
We established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell-based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.
... Furthermore, the strong fragmentation and the short sizes of circulating DNA fragments do not allow the direct detection of these mutations. An alternative approach is to perform analysis on circulating fetal trophoblastic cells (CFTCs) isolated from the maternal blood [17]. ...
Huntington’s disease (HD) is an autosomal dominant disease that immensely impacts the affected families. However, the transmission of the disease from carriers to their offspring could be prevented. Prenatal diagnosis (PND) and preimplantation genetic diagnosis (PGD) are the only two available reproductive options for the carriers at risk to have disease-free children. PND for HD could be done through two kinds of genetic testing: direct and indirect. The same approaches are available for preimplantation genetic testing. In addition, a third alternative is nondisclosure testing, which is only available in the case of PGD. The pros and cons of different approaches are discussed. However, only a relatively few at-risk parents opt for PND and PGD. Furthermore, compared to PND, PGD is even more seldom opted for as a reproductive option.
