Cell-based Noninvasive Prenatal Testing Research Articles

Clinical interpretation of cell-based non-invasive prenatal testing for monogenic disorders including repeat expansion disorders: potentials and pitfalls.

Introduction: Circulating fetal cells isolated from maternal blood can be used for prenatal testing, representing a safe alternative to invasive testing. The present study investigated the potential of cell-based noninvasive prenatal testing (NIPT) for diagnosing monogenic disorders dependent on the mode of inheritance. Methods: Maternal blood samples were collected from women opting for prenatal diagnostics for specific monogenic disorders (N = 7). Fetal trophoblasts were enriched and stained using magnetic activated cell sorting and isolated by fluorescens activated single-cell sorting. Individual cells were subject to whole genome amplification, and cells of fetal origin were identified by DNA-profiling using short tandem repeat markers. The amplified fetal DNA was input for genetic testing for autosomal dominant-, autosomal recessive-, X-linked and repeat expansion disorders by direct variant analysis and haplotyping. The cell-based NIPT results were compared with those of invasive testing. Results: In two cases at risk of skeletal dysplasia, caused by variants in the FGFR3 gene (autosomal dominant disorders), cell-based NIPT correctly stated an affected fetus, but allelic dropout of the normal alleles were observed in both cases. Cell-based NIPT gave an accurate result in two cases at risk of autosomal recessive disorders, where the parents carried either different diastrophic dysplasia causing variants in the SLC26A2 gene or the same cystic fibrosis disease-causing variant in the CFTR gene. Cell-based NIPT accurately identified an affected male fetus in a pregnancy at risk of Duchenne muscular dystrophy (DMD gene, X-linked recessive disorders). In two cases at risk of the myotonic dystrophy type 1 (DMPK gene, repeat expansion disorder), cell-based NIPT correctly detected an affected and an unaffected fetus, respectively. Discussion: Circulating fetal cells can be used to detect both maternally- and paternally inherited monogenic disorders irrespective of the type of variant, however, the risk of allelic dropout must be considered. We conclude that the clinical interpretation of the cell-based NIPT result thus varies depending on the disorders' mode of inheritance.

Single-cell whole-genome sequencing, haplotype analysis in prenatal diagnosis of monogenic diseases.

Monogenic inherited diseases are common causes of congenital disabilities, leading to severe economic and mental burdens on affected families. In our previous study, we demonstrated the validity of cell-based noninvasive prenatal testing (cbNIPT) in prenatal diagnosis by single-cell targeted sequencing. The present research further explored the feasibility of single-cell whole-genome sequencing (WGS) and haplotype analysis of various monogenic diseases with cbNIPT. Four families were recruited: one with inherited deafness, one with hemophilia, one with large vestibular aqueduct syndrome (LVAS), and one with no disease. Circulating trophoblast cells (cTBs) were obtained from maternal blood and analyzed by single-cell 15X WGS. Haplotype analysis showed that CFC178 (deafness family), CFC616 (hemophilia family), and CFC111 (LVAS family) inherited haplotypes from paternal and/or maternal pathogenic loci. Amniotic fluid or fetal villi samples from the deafness and hemophilia families confirmed these results. WGS performed better than targeted sequencing in genome coverage, allele dropout (ADO), and false-positive (FP) ratios. Our findings suggest that cbNIPT by WGS and haplotype analysis have great potential for use in prenatally diagnosing various monogenic diseases.

Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing-based non-invasive genetic testing.

PurposeAlthough 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.MethodsUsing 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.ResultsThe 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.ConclusionsWe 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.

HUNTER-Chip: Bioinspired Hierarchically Aptamer Structure-Based Circulating Fetal Cell Isolation for Non-Invasive Prenatal Testing.

Isolation and genetic analysis of circulating fetal cells from billions of maternal cells in peripheral blood are the cornerstone of fetal cell-based non-invasive prenatal testing. Inspired by the hierarchically multivalent architecture for enhanced capture of nature, an aptamer-based Hierarchically mUltivalent aNTibody mimic intERface (HUNTER) was designed with a tremendous avidity effect for highly efficient capture and non-destructive release of fetal cells. It was engineered by grafting Y-shaped DNA nanostructures to a linear polymer chain, creating a flexible polymer chain with bivalent aptamer side chains. This hierarchical arrangement of the aptamer ensures morphological complementarity, collective multiple-site interaction, and multivalent recognition between the aptamer and target cells. In combination with a deterministic lateral displacement (DLD)-patterned microdevice named as HUNTER-Chip, it achieves a binding affinity over 65-fold and a capture efficiency over 260%-fold due to the combination of hierarchically designed aptamers and frequent cell-ligand collision created by DLD. Moreover, a nuclease-assisted cell release strategy facilitates the release of fetal cells for gene analysis, such as fluorescence in situ hybridization. With the advantages of high affinity, excellent capture efficiency, and compatible downstream analysis, the HUNTER-Chip holds great potential for non-invasive prenatal diagnosis.

Use of amplicon-based sequencing for testing fetal identity and monogenic traits with Single Circulating Trophoblast (SCT) as one form of cell-based NIPT.

A major challenge for cell-based non-invasive prenatal testing (NIPT) is to distinguish individual presumptive fetal cells from maternal cells in female pregnancies. We have sought a rapid, robust, versatile, and low-cost next-generation sequencing method to facilitate this process. Toward this goal, single isolated cells underwent whole genome amplification prior to genotyping. Multiple highly polymorphic genomic regions (including HLA-A and HLA-B) with 10-20 very informative single nucleotide polymorphisms (SNPs) within a 200 bp interval were amplified with a modified method based on other publications. To enhance the power of cell identification, approximately 40 Human Identification SNP (Applied Biosystems) test amplicons were also utilized. Using SNP results to compare to sex chromosome data from NGS as a reliable standard, the true positive rate for genotyping was 83.4%, true negative 6.6%, false positive 3.3%, and false negative 6.6%. These results would not be sufficient for clinical diagnosis, but they demonstrate the general validity of the approach and suggest that deeper genotyping of single cells could be completely reliable. A paternal DNA sample is not required using this method. The assay also successfully detected pathogenic variants causing Tay Sachs disease, cystic fibrosis, and hemoglobinopathies in single lymphoblastoid cells, and disease-causing variants in three cell-based NIPT cases. This method could be applicable for any monogenic diagnosis.

Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing

PurposeProof 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).MethodPGT-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.ResultsEight 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.ConclusionThese 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 numberN-20180001

Kinship analysis on single cells after whole genome amplification

Short Tandem Repeat (STR-) and Single Nucleotide Polymorphism (SNP-) genotyping have been extensively studied within forensic kinship analysis. Nevertheless, no results have been reported on kinship analysis after whole genome amplification (WGA) of single cells. This WGA step is a necessary procedure in several applications, such as cell-based non-invasive prenatal testing (cbNIPT) and pre-implantation genetic diagnosis (PGD). In cbNIPT, all putative fetal cells must be discriminated from maternal cells after enrichment from whole blood. This study investigates the efficacy and evidential value of STR- and SNP-genotyping methods for the discrimination of 24 single cells after WGA, within three families. Formaldehyde-fixed and unfixed cells are assessed in offspring-parent duos and offspring-mother-father trios. Results demonstrate that both genotyping methods can be used in all tested conditions and scenarios with 100% sensitivity and 100% specificity, with a similar evidential value for fixed and unfixed cells. Moreover, sequence-based SNP-genotyping results in a higher evidential value than length-based STR-genotyping after WGA, which is not observed using high-quality offspring bulk DNA samples. Finally, it is also demonstrated that the availability of the DNA genotypes of both parents strongly increases the evidential value of the results.

The Number of Circulating Fetal Extravillous Trophoblasts Varies from Gestational Week 6 to 20

Cell-based non-invasive prenatal testing (cbNIPT) based on circulating fetal extravillous trophoblasts (fEVTs) has shown to be possible in gestational week (GW) 10–13. Prenatal testing is relevant for a wider time period than GW 10–13, but it is unclear if fEVTs are present in sufficient numbers for cbNIPT at other time points during pregnancy. We present the first longitudinal study where the number of circulating fEVTs was determined from the mid first trimester to the mid second, specifically GW 6–8, 12–13, and 19–20. Blood samples from 13 women opting for assisted reproduction were collected at GW 6–8, 12–13, and 19–20. fEVTs were enriched using a magnetic-activated cell sorting system, stained with anti-cytokeratin antibodies, and fEVTs were identified with the use of a MetaSystem fluorescence microscope scanner. Blood samples drawn at GW 6–8 yielded an average of 5.5 fEVTs per 30 mL of blood. This increased significantly to an average of 11.8 in GW 12–13 (P value: 0.0070, Mann-Whitney test), and decreased significantly to an average of 5.3 in GW 19–20 (P value: 0.0063, Mann-Whitney test). In 9 out of 13 cases, the number of fEVTs peaked in GW 12–13 compared to GW 6–8 and GW 19–20. For the majority of cases, fEVTs can be identified at GW 6–8 and GW 19–20, but the highest number of fEVTs is observed at GW 12–13 indicating this is the optimal time point for cbNIPT.

Stimuli-Responsive Microfluidic Interface Enables Highly Efficient Capture and Release of Circulating Fetal Cells for Non-Invasive Prenatal Testing.

Circulating fetal nucleated cells (CFCs) carrying whole genomic coding of the fetus in maternal blood have been pursued as ideal biomarkers for noninvasive prenatal testing (NIPT). However, a significant limitation is the need to enrich sufficient cells in quantity and purity for fetal genetic disorder diagnosis. This study for the first time demonstrates a stimuli-responsive ligand enabling interface on array patterned microfluidic chip (NIPT-Chip) for high efficient isolation and release of CFCs in untreated whole blood. Deterministic lateral displacement (DLD)-array was patterned in the chip to increase collision frequency between CFCs and surface-anchored antibody to achieve high efficient cell capture. More importantly, the stimuli-responsive interface enables gentle release of captured CFCs through a thiol exchange reaction for downstream gene analysis of NIPT. With the advantages of simple processing, efficient isolation, and gentle release, NIPT-Chip offers great potential for clinical translation of circulating fetal cell-based NIPT.

Do fetal extravillous trophoblasts circulate in maternal blood postpartum?

IntroductionCirculating fetal extravillous trophoblasts may offer a superior alternative to cell‐free fetal DNA for noninvasive prenatal testing. Cells of fetal origin are a pure source of fetal genome; hence, unlike the cell‐free noninvasive prenatal test, the fetal cell‐based noninvasive prenatal test is not expected to be affected by maternal DNA. However, circulating fetal cells from previous pregnancies may lead to confounding results.Material and methodsTo study whether fetal trophoblast cells persist in maternal circulation postpartum, blood samples were collected from 11 women who had given birth to a boy, with blood sampling at 1‐3 days (W0), 4‐5 weeks (W4‐5), around 8 weeks (W8) and around 12 weeks (W12) postpartum. The existence of fetal extravillous trophoblasts was verified either by X and Y chromosome fluorescence in situ hybridization analysis or by short tandem repeat analysis. To exclude technological bias in isolating fetal cells, blood samples were also collected from 10 pregnant women between a gestational age of 10 and 14 weeks, the optimal time frame for cell‐based noninvasive prenatal test sampling. All the samples were processed according to protocols established by ARCEDI Biotech for fetal extravillous trophoblast enrichment and isolation.ResultsFetal extravillous trophoblasts were found in all the 10 samples from pregnant women between a gestational age of 10 and 14 weeks. However, only 4 of 11 blood samples taken from women at 1‐3 days postpartum rendered fetal extravillous trophoblasts, and only 2 of 11 samples rendered fetal extravillous trophoblasts at 4 weeks postpartum.ConclusionsIn this preliminary dataset on few pregnancies, none of the samples rendered any fetal cells at or after 8 weeks postpartum, showing that cell‐based noninvasive prenatal testing based on fetal extravillous trophoblasts is unlikely to be influenced by circulating cells from previous pregnancies.

900: A pilot validation study for cell-based noninvasive prenatal testing (NIPT) in 42 cases

900: A pilot validation study for cell-based noninvasive prenatal testing (NIPT) in 42 cases

Reliable detection of subchromosomal deletions and duplications using cell-based noninvasive prenatal testing.

ObjectiveTo gather additional data on the ability to detect subchromosomal abnormalities of various sizes in single fetal cells isolated from maternal blood, using low‐coverage shotgun next‐generation sequencing for cell‐based noninvasive prenatal testing (NIPT).MethodFetal trophoblasts were recovered from approximately 30 mL of maternal blood using maternal white blood cell depletion, density‐based cell separation, immunofluorescence staining, and high‐resolution scanning. These trophoblastic cells were picked as single cells and underwent whole genome amplification for subsequent genome‐wide copy number analysis and genotyping to confirm the fetal origin of the cells.ResultsApplying our fetal cell isolation method to a series of 125 maternal blood samples, we detected on average 4.17 putative fetal cells/sample. The series included 15 cases with clinically diagnosed fetal aneuploidies and five cases with subchromosomal abnormalities. This method was capable of detecting findings that were 1 to 2 Mb in size, and all were concordant with the microarray or karyotype data obtained on a fetal sample. A minority of fetal cells showed evidence of genome degradation likely related to apoptosis.ConclusionWe demonstrate that this cell‐based NIPT method has the capacity to reliably diagnose fetal chromosomal abnormalities down to 1 to 2 Mb in size.

STR profiling and Copy Number Variation analysis on single, preserved cells using current Whole Genome Amplification methods

The growing interest in liquid biopsies for cancer research and cell-based non-invasive prenatal testing (NIPT) invigorates the need for improved single cell analysis. In these applications, target cells are extremely rare and fragile in peripheral circulation, which makes the genetic analysis very challenging. To overcome these challenges, cell stabilization and unbiased whole genome amplification are required. This study investigates the performance of four WGA methods on single or a limited number of cells after 24 hour of Streck Cell-Free DNA BCT preservation. The suitability of the DNA, amplified with Ampli1, DOPlify, PicoPLEX and REPLI-g, was assessed for both short tandem repeat (STR) profiling and copy number variant (CNV) analysis after shallow whole genome massively parallel sequencing (MPS). Results demonstrate that Ampli1, DOPlify and PicoPLEX perform well for both applications, with some differences between the methods. Samples amplified with REPLI-g did not result in suitable STR or CNV profiles, indicating that this WGA method is not able to generate high quality DNA after Streck Cell-Free DNA BCT stabilization of the cells.

Evidence for Feasibility of Fetal Trophoblastic Cell-Based Noninvasive Prenatal Testing

(Abstracted from Prenat Diagn 2016;36:1–11) The potential use of fetal cells in maternal blood for prenatal diagnosis has been a promising technology for decades. Several strategies have been tried and tested for recovering fetal cells, although they are inconsistent and not reproducible.

Genome-wide copy number analysis on DNA from fetal cells isolated from the blood of pregnant women.

Non-invasive prenatal testing (NIPT) based on fetal cells in maternal blood has the advantage over NIPT based on circulating cell-free fetal DNA in that there is no contamination with maternal DNA. This will most likely result in better detection of chromosomal aberrations including subchromosomal defects. The objective of this study was to test whether fetal cells enriched from maternal blood can be used for cell-based NIPT. We present a method for enriching fetal cells from maternal blood, subsequent amplification of the fetal genome and detection of chromosomal and subchromosomal variations in the genome. An average of 12.8 fetal cells from 30 mL of maternal blood were recovered using our method. Subsequently, whole genome amplification on fetal cells resulted in amplified fetal DNA in amounts and quality high enough to generate array comparative genomic hybridization as well as next-generation sequencing profiles. From one to two fetal cells, we were able to demonstrate copy number differences of whole chromosomes (21, X-, and Y) as well as subchromosomal aberrations (ring X). Intact fetal cells can be isolated from every maternal blood sample. Amplified DNA from isolated fetal cells enabled genetic analysis by array comparative genomic hybridization and next-generation sequencing. © 2016 John Wiley & Sons, Ltd.