A Novel Method to Identify Syncytiotrophoblast-Derived RNA Products Representative of Trisomy 21 Placental RNA in Maternal Plasma

2008 ◽  
pp. 291-302 ◽  
Author(s):  
Attie T. J. J. Go ◽  
Allerdien Visser ◽  
Marie Dijk ◽  
Monique A. M. Mulders ◽  
Paul Eijk ◽  
...  
Keyword(s):  
Trisomy 21 ◽  
Maternal Plasma ◽  
Novel Method

Non-invasive prenatal detection of trisomy 21 by quantifying segmental duplication in maternal plasma with digital PCR

2016 ◽  
Vol 8 (10) ◽  
pp. 2138-2143 ◽  
Author(s):  
Shu Xu ◽  
Bingjie Zou ◽  
Zheng Xiang ◽  
Mingzhu Miao ◽  
Qinxin Song ◽  
...  
Keyword(s):  
Segmental Duplication ◽  
Trisomy 21 ◽  
Digital Pcr ◽  
Maternal Plasma ◽  
Chromosome 21 ◽  
Non Invasive ◽  
Prenatal Detection ◽  
Novel Method

We proposed a novel method for the detection of trisomy 21 by quantifying the slightly increased amount of chromosome 21 in cfDNA.

Maternal plasma phospholipids are altered in trisomy 21 cases and prior to preeclampsia and preterm outcomes

2014 ◽  
Vol 28 (14) ◽  
pp. 1635-1638 ◽  
Author(s):  
Joana Pinto ◽  
Elisabete Maciel ◽  
Tânia S. Melo ◽  
M. Rosário M. Domingues ◽  
Eulália Galhano ◽  
...  
Keyword(s):  
Trisomy 21 ◽  
Maternal Plasma ◽  
Plasma Phospholipids

scholarly journals Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex

2016 ◽  
Vol 62 (6) ◽  
pp. 848-855 ◽  
Author(s):  
George Koumbaris ◽  
Elena Kypri ◽  
Kyriakos Tsangaras ◽  
Achilleas Achilleos ◽  
Petros Mina ◽  
...  
Keyword(s):  
Trisomy 21 ◽  
Dna Analysis ◽  
Prenatal Testing ◽  
Cost Effective ◽  
Maternal Plasma ◽  
Trisomy 13 ◽  
Cell Free Dna ◽  
Free Dna ◽  
Genomic Regions ◽  
Fetal Fraction

Abstract BACKGROUND There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%–100%) cases of trisomy 21, 16/16 (95% CI, 79.4%–100%) cases of trisomy 18, 5/5 (95% CI, 47.8%–100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%–100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%–100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

scholarly journals Measurement of Allelic-Expression Ratios in Trisomy 21 Placentas by Quencher Extension of Heterozygous Samples Identified by Partially Denaturing HPLC

2008 ◽  
Vol 54 (2) ◽  
pp. 437-440 ◽  
Author(s):  
Attie T J I Go ◽  
Allerdien Visser ◽  
Ofir T Betsalel ◽  
John M G van Vugt ◽  
Marinus A Blankenstein ◽  
...  
Keyword(s):  
Real Time ◽  
Trisomy 21 ◽  
First Trimester ◽  
Maternal Plasma ◽  
Rapid Identification ◽  
Chromosome 21 ◽  
Wave System ◽  
Exon 2 ◽  
Reading Frame ◽  
Time Required

Abstract Background: Measuring the allelic ratios of placental transcripts in maternal plasma permits noninvasive prenatal detection of chromosomal aneuploidy. Current methods, however, require highly specialized equipment (MALDI-TOF), limiting the widespread implementation of this powerful RNA single-nucleotide polymorphism (SNP) strategy in routine diagnostic settings. We adapted and applied the Transgenomic WAVE System and quencher extension (QEXT) for this purpose. Methods: The expressed SNP (rs2187247) in exon 2 of the placentally expressed C21orf105 gene (chromosome 21 open reading frame 105) on chromosome 21 was tested in a trisomy 21 model system in which we obtained RNA selectively released from syncytiotrophoblasts of normal and trisomy 21 placentas during first trimester. Results: In identifying heterozygous samples, we observed an exact correspondence between sequencing results and results obtained with the WAVE System. With respect to the analysis time required, the WAVE System was superior. In addition, the real-time QEXT assay (as optimized and validated with calibration standards consisting of 262-bp C21orf105 cDNA amplicons) accurately measured allele ratios after we optimized fragment purification, concentrations of input DNA and quencher label, and calculations of reporter signals. Finally, the optimized and validated QEXT assay correctly distinguished normal placentas from trisomy 21 placentas in tests of the following clinically relevant combinations: diploid homozygous (CC), diploid heterozygous (AC), triploid homozygous (AAA), and triploid heterozygous (AAC or ACC). Conclusion: The QEXT method, which is directly adaptable to current real-time PCR equipment, along with rapid identification of informative samples with the WAVE System, may facilitate routine implementation of the RNA-SNP assay for noninvasive aneuploidy diagnostics.

scholarly journals Noninvasive Prenatal Detection of Trisomy 21 by an Epigenetic–Genetic Chromosome-Dosage Approach

2010 ◽  
Vol 56 (1) ◽  
pp. 90-98 ◽  
Author(s):  
Yu K Tong ◽  
Shengnan Jin ◽  
Rossa WK Chiu ◽  
Chunming Ding ◽  
KC Allen Chan ◽  
...  
Keyword(s):  
Prenatal Diagnosis ◽  
Genetic Markers ◽  
Blood Cells ◽  
Trisomy 21 ◽  
Maternal Blood ◽  
Maternal Plasma ◽  
Plasma Samples ◽  
Prenatal Detection ◽  
Fetal Dna ◽  
Noninvasive Prenatal Diagnosis

Abstract Background: The use of fetal DNA in maternal plasma for noninvasive prenatal diagnosis of trisomy 21 (T21) is an actively researched area. We propose a novel method of T21 detection that combines fetal-specific epigenetic and genetic markers. Methods: We used combined bisulfite restriction analysis to search for fetal DNA markers on chromosome 21 that were differentially methylated in the placenta and maternal blood cells and confirmed any target locus with bisulfite sequencing. We then used methylation-sensitive restriction endonuclease digestion followed by microfluidics digital PCR analysis to investigate the identified marker. Chromosome-dosage analysis was performed by comparing the dosage of this epigenetic marker with that of the ZFY (zinc finger protein, Y-linked) gene on chromosome Y. Results: The putative promoter of the HLCS (holocarboxylase synthetase) gene was hypermethylated in the placenta and hypomethylated in maternal blood cells. A chromosome-dosage comparison of the hypermethylated HLCS and ZFY loci could distinguish samples of T21 and euploid placental DNA. Twenty-four maternal plasma samples from euploid pregnancies and 5 maternal plasma samples from T21 pregnancies were analyzed. All but 1 of the euploid samples were correctly classified. Conclusions: The epigenetic–genetic chromosome-dosage approach is a new method for noninvasive prenatal detection of T21. The epigenetic part of the analysis can be applied to all pregnancies. Because the genetic part of the analysis uses paternally inherited, fetal-specific genetic markers that are abundant in the genome, broad population coverage should be readily achievable. This approach has the potential to become a generally usable technique for noninvasive prenatal diagnosis.

scholarly journals Synergy of Total PLAC4 RNA Concentration and Measurement of the RNA Single-Nucleotide Polymorphism Allelic Ratio for the Noninvasive Prenatal Detection of Trisomy 21

2010 ◽  
Vol 56 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Nancy BY Tsui ◽  
Ranjit Akolekar ◽  
Rossa WK Chiu ◽  
Katherine CK Chow ◽  
Tak Y Leung ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Real Time Pcr ◽  
Trisomy 21 ◽  
Digital Pcr ◽  
Maternal Plasma ◽  
Chromosome 21 ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Allelic Ratio ◽  
Mrna Concentration

Abstract Background: Maternal plasma mRNA encoded by the PLAC4 gene (placenta-specific 4), which is transcribed from chromosome 21 in placental cells, is a potential marker for the noninvasive assessment of chromosome 21 dosage in the fetus. We evaluated the diagnostic sensitivities and specificities of 2 trisomy 21–screening approaches that use maternal plasma PLAC4 mRNA. Methods: We studied maternal plasma samples from 153 pregnant women carrying euploid and trisomy 21 fetuses. For the samples in which the fetuses were heterozygous for the studied PLAC4 single-nucleotide polymorphism (SNP), we measured the ratio between 2 alleles of the SNP in maternal plasma PLAC4 mRNA (RNA-SNP) by mass spectrometric (MS) and digital PCR methods. For pregnancies involving fetuses homozygous for the SNP, we quantified the total PLAC4 mRNA concentration in maternal plasma by real-time PCR and digital PCR. Results: For the RNA-SNP approach, we achieved a diagnostic sensitivity and specificity of 100% (95% CI, 40.2%–100%) and 89.7% (95% CI, 78.8%–96.1%), respectively, for both the MS and the digital PCR methods. For the mRNA-quantification approach, the areas under the ROC curves were 0.859 (95% CI, 0.741–0.903) and 0.833 (95% CI, 0.770–0.923) for plasma PLAC4 mRNA concentrations measured by the real-time PCR and the digital PCR methods, respectively. Conclusions: For prenatal screening of trisomy 21, the quantification of the total PLAC4 mRNA concentration can be used in a synergistic manner with the RNA-SNP allelic ratio approach to increase the population coverage of cases in which diagnostic information can be obtained.

scholarly journals Increased Fetal DNA Concentrations in the Plasma of Pregnant Women Carrying Fetuses with Trisomy 21

1999 ◽  
Vol 45 (10) ◽  
pp. 1747-1751 ◽  
Author(s):  
YM Dennis Lo ◽  
Tze K Lau ◽  
Jun Zhang ◽  
Tse N Leung ◽  
Allan MZ Chang ◽  
...  
Keyword(s):  
Hong Kong ◽  
Real Time ◽  
Trisomy 21 ◽  
Clinical Investigation ◽  
Maternal Plasma ◽  
Pcr Analysis ◽  
Fetal Dna ◽  
Prenatal Diagnostic ◽  
Cell Free Fetal Dna ◽  
Circulating Fetal Dna

Abstract Background: The recent discovery of the presence of circulating cell-free fetal DNA in maternal plasma opens up new prenatal diagnostic applications and provides new avenues for clinical investigation. It is of research and potential diagnostic interest to determine whether fetal trisomy 21 may be associated with quantitative abnormalities of circulating fetal DNA in maternal plasma. Methods: Maternal plasma samples were prospectively collected from two centers situated in Hong Kong and Boston. Samples collected from Boston consisted of 7 women carrying male trisomy 21 fetuses, 19 carrying euploid male fetuses, and 13 carrying female fetuses. Samples collected from Hong Kong consisted of 6 women carrying male trisomy 21 fetuses, 18 carrying euploid male fetuses, and 10 carrying female fetuses. Male fetal DNA in maternal plasma was measured using real-time quantitative Y-chromosomal PCR. Results: For patients recruited from Boston, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 46.0 genome-equivalents/mL and 23.3 genome-equivalents/mL, respectively (P = 0.028). For patients recruited from Hong Kong, the median circulating fetal DNA concentrations in women carrying trisomy 21 and euploid male fetuses were 48.2 genome-equivalents/mL and 16.3 genome-equivalents/mL, respectively (P = 0.026). None of the samples from women carrying female fetuses had detectable Y-chromosomal signals. Conclusions: Abnormally high concentrations of circulating fetal DNA are found in a proportion of women carrying fetuses with trisomy 21. The robustness and reproducibility of real-time PCR analysis of maternal plasma makes it a valuable tool for cross-institutional collaboration involving centers located in different parts of the world.

scholarly journals Single Molecule Sequencing of Free DNA from Maternal Plasma for Noninvasive Trisomy 21 Detection

2012 ◽  
Vol 58 (4) ◽  
pp. 699-706 ◽  
Author(s):  
Jessica M E van den Oever ◽  
Sahila Balkassmi ◽  
E Joanne Verweij ◽  
Maarten van Iterson ◽  
Phebe N Adama van Scheltema ◽  
...  
Keyword(s):  
Single Molecule ◽  
Trisomy 21 ◽  
Sequence Data ◽  
Pcr Amplification ◽  
Maternal Plasma ◽  
Single Molecule Sequencing ◽  
Experimental Noise ◽  
Fetal Aneuploidy ◽  
Free Dna ◽  
Gc Bias

Abstract BACKGROUND Noninvasive fetal aneuploidy detection by use of free DNA from maternal plasma has recently been shown to be achievable by whole genome shotgun sequencing. The high-throughput next-generation sequencing platforms previously tested use a PCR step during sample preparation, which results in amplification bias in GC-rich areas of the human genome. To eliminate this bias, and thereby experimental noise, we have used single molecule sequencing as an alternative method. METHODS For noninvasive trisomy 21 detection, we performed single molecule sequencing on the Helicos platform using free DNA isolated from maternal plasma from 9 weeks of gestation onwards. Relative sequence tag density ratios were calculated and results were directly compared to the previously described Illumina GAII platform. RESULTS Sequence data generated without an amplification step show no GC bias. Therefore, with the use of single molecule sequencing all trisomy 21 fetuses could be distinguished more clearly from euploid fetuses. CONCLUSIONS This study shows for the first time that single molecule sequencing is an attractive and easy to use alternative for reliable noninvasive fetal aneuploidy detection in diagnostics. With this approach, previously described experimental noise associated with PCR amplification, such as GC bias, can be overcome.

Sign in / Sign up

Export Citation Format

Share Document