Dataset Information

Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

ABSTRACT: OBJECTIVE:NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles. METHOD:After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of plasma DNA from 213 pregnant women. Molecular counting of circulating cell-free DNA was performed using a mix of hydrolysis probes targeting chromosome 21 and a reference chromosome. RESULTS:The results of our validation experiments showed that ddPCR detected trisomy 21 even when the sample's trisomic DNA content is as low as 5%. In a validation study of plasma samples from 213 pregnant women, ddPCR discriminated clearly between the trisomy 21 and the euploidy groups. CONCLUSION:Our results demonstrate that digital PCR can meet the requirements for non-invasive prenatal testing of trisomy 21. This approach is technically simple, relatively cheap, easy to implement in a diagnostic setting and compatible with ethical concerns regarding access to nucleotide sequence information. These advantages make it a potential technique of choice for population-wide screening for trisomy 21 in pregnant women.

SUBMITTER: El Khattabi LA

PROVIDER: S-EPMC4864235 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

El Khattabi Laïla Allach LA Rouillac-Le Sciellour Christelle C Le Tessier Dominique D Luscan Armelle A Coustier Audrey A Porcher Raphael R Bhouri Rakia R Nectoux Juliette J Sérazin Valérie V Quibel Thibaut T Mandelbrot Laurent L Tsatsaris Vassilis V Vialard François F Dupont Jean-Michel JM

PloS one 20160511 5

<h4>Objective</h4>NIPT for fetal aneuploidy by digital PCR has been hampered by the large number of PCR reactions needed to meet statistical requirements, preventing clinical application. Here, we designed an octoplex droplet digital PCR (ddPCR) assay which allows increasing the number of available targets and thus overcomes statistical obstacles.<h4>Method</h4>After technical optimization of the multiplex PCR on mixtures of trisomic and euploid DNA, we performed a validation study on samples of ...[more]

PMID: 27167625

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Project description:BACKGROUND: Screening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA. METHODS AND FINDINGS: Highly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects. CONCLUSIONS: In summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma.

Project description:ObjectivesTo validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling.DesignDiagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples.SettingPrenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands.Participants753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing.Main outcome measuresProportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection.ResultsResults were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%.ConclusionMultiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

Project description:A universal biomarker panel with the potential to predict high-risk pregnancies or adverse pregnancy outcome does not exist. Transcriptome analysis is a powerful tool to capture differentially expressed genes (DEG), which can be used as biomarker-diagnostic-predictive tool for various conditions in prenatal setting. In search of biomarker set for predicting high-risk pregnancies, we performed global expression profiling to find DEG in Ts21. Subsequently, we performed targeted validation and diagnostic performance evaluation on a larger group of case and control samples. Initially, transcriptomic profiles of 10 cultivated amniocyte samples with Ts21 and 9 with normal euploid constitution were determined using expression microarrays. Datasets from Ts21 transcriptomic studies from GEO repository were incorporated. DEG were discovered using linear regression modelling and validated using RT-PCR quantification on an independent sample of 16 cases with Ts21 and 32 controls. The classification performance of Ts21 status based on expression profiling was performed using supervised machine learning algorithm and evaluated using a leave-one-out cross validation approach. Global gene expression profiling has revealed significant expression changes between normal and Ts21 samples, which in combination with data from previously performed Ts21 transcriptomic studies, were used to generate a multi-gene biomarker for Ts21, comprising of 9 gene expression profiles. In addition to biomarker's high performance in discriminating samples from global expression profiling, we were also able to show its discriminatory performance on a larger sample set 2, validated using RT-PCR experiment (AUC=0.97), while its performance on data from previously published studies reached discriminatory AUC values of 1.00. Our results show that transcriptomic changes might potentially be used to discriminate trisomy of chromosome 21 in the prenatal setting. As expressional alterations reflect both, causal and reactive cellular mechanisms, transcriptomic changes may thus have future potential in the diagnosis of a wide array of heterogeneous diseases that result from genetic disturbances.

Project description:Objective: Cell-free fetal DNA (cffDNA) testing has opened new options in prenatal screening for trisomy 21. Due to the higher costs of cffDNA testing there is an ongoing debate on how to combine different screening strategies. Methods: For this study, a model-based approach was used to evaluate all births in Germany in 2012 together with the percentage of euploid and trisomic pregnancies. Detection rates (DR), false positive rates (FPR), the costs of different screening strategies for trisomy 21 and combinations of these strategies were compared. The number of fetuses with trisomy 21 at 12 + 0 weeks of gestation was estimated based on maternal age distribution. We examined the screening performance of a screening strategy based on maternal age, first trimester screening (FTS) and cffDNA testing as well as the combinations "maternal age and cffDNA" and "FTS and cffDNA". Results: In 2012 673 544 children were born. Median maternal age at delivery was 30.2 years (25th-75th quartile: 27.0-34.0). Based on maternal age distribution the expected number of fetuses with trisomy 21 at 12 weeks' gestation was 1788. Our study population therefore consisted of 675 332 pregnancies. Screening based only on maternal age or FTS or cffDNA resulted in detection rates of 63.3 %, 92.2 % and 99.0 % and false positive rates of 21.8 %, 8.0 % and 0.1 %, respectively. When maternal age was combined with cffDNA, cffDNA testing was only offered to women over a certain age; if a cut-off of 30 years was used, this resulted in a DR of 85.2 % and a FPR of 1.7 %. If primary screening consisted of FTS with cffDNA testing only done when the risk was between 1 : 10 and 1 : 1000, the detection rate was 96.7 % and the false positive rate was 1.2 %. Conclusion: In this model-based study we showed that prenatal screening for trisomy 21 can be improved even more by combining FTS and cffDNA. Further studies are necessary to examine whether these results can be reproduced in reality.

Dataset Information

Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

Publications

Could Digital PCR Be an Alternative as a Non-Invasive Prenatal Test for Trisomy 21: A Proof of Concept Study.

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