A genome-wide search for new imprinted genes in the human placenta identifies DSCAM as the first imprinted gene on chromosome 21

Notice à reprendre en chantier qualité avec la version finale de l’EditeurInternational audienceWe identified, through a genome-wide search for new imprinted genes in the human placenta, DSCAM (Down Syndrome Cellular Adhesion Molecule) as a paternally expressed imprinted gene. Our work revealed the presence of a Differentially Methylated Region (DMR), located within intron 1 that might regulate the imprinting in the region. This DMR showed a maternal allele methylation, compatible with its paternal expression. We showed that DSCAM is present in endothelial cells and the syncytiotrophoblast layer of the human placenta. In mouse, Dscam expression is biallelic in foetal brain and placenta excluding any possible imprinting in these tissues. This gene encodes a cellular adhesion molecule mainly known for its role in neurone development but its function in the placenta remains unclear. We report here the first imprinted gene located on human chromosome 21 with potential clinical implications

Droplet Digital PCR combined with minisequencing, a new approach to analyze fetal DNA from maternal blood: application to the non-invasive prenatal diagnosis of achondroplasia.

International audienceBackground - Achondroplasia is generally detected by abnormal prenatal ultrasound findings in the third trimester of pregnancy and then confirmed by molecular genetic testing of fetal genomic DNA obtained by aspiration of amniotic fluid. This invasive procedure presents a small but significant risk for both the fetus and mother. Therefore, non-invasive procedures using cell-free fetal DNA in maternal plasma have been developed for the detection of the fetal achondroplasia mutations. Methods - To determine whether the fetus carries the de novo mis-sense genetic mutation at nucleotide 1138 in FGFR3 gene involved in >99% of achondroplasia cases, we developed two independent methods: digital-droplet PCR combined with minisequencing, which are very sensitive methods allowing detection of rare alleles. Results - We collected 26 plasmatic samples from women carrying fetus at risk of achondroplasia and diagnosed to date a total of five affected fetuses in maternal blood. The sensitivity and specificity of our test are respectively 100% [95% confidence interval, 56.6-100%] and 100% [95% confidence interval, 84.5-100%]. Conclusions - This novel, original strategy for non-invasive prenatal diagnosis of achondroplasia is suitable for implementation in routine clinical testing and allows considering extending the applications of these technologies in non-invasive prenatal diagnosis of many other monogenic diseases. © 2016 John Wiley & Sons, Ltd

Performance of Semiconductor sequencing platform for non-invasive prenatal genetic screening for fetal aneuploidies: results from a multicenter prospective cohort study in a clinical setting

Objectives To validate and evaluate an integrated protocol for non‐invasive prenatal genetic screening (NIPS) for common fetal aneuploidies in a clinical setting, using the semiconductor sequencing technology, Ion Proton. Methods This prospective cohort study included 2505 pregnant women from seven academic genetic laboratories (695 high risk pregnancies in a validation study and 1810 pregnancies with a risk higher than 1/250 without ultrasound anomalies, in a real NIPS clinical setting). Cell free DNA from plasma samples was sequenced using Ion Proton sequencer, and sequencing data were analyzed using the open‐access software WISECONDOR. Performance metrics for detection of trisomies 21, 18 and 13, were calculated based on either fetal karyotype result or clinical data collected at birth. We also evaluated the failure rate and compared three methods of fetal fraction quantification (RASSF1A assay, DEFRAG and SANEFALCON software). Results Sensitivities and specificities were: 98.3% (95%CI: 93.5 ‐ 99.7) and 99.9% (95%CI: 99.4 ‐ 100) for T21, 96.7% (95%CI: 80.9 ‐ 99.8) and 100% (95%CI: 99.6 ‐ 100) for T18, 94.1% (95%CI: 69.2 ‐ 99.7) and 100% (95%CI: 99.6 ‐ 100) for T13. Our failure rate was 1.2% at first and as low as 0.6% after re‐testing some of the failed samples. Fetal fraction estimation by RASSF1A assay was consistent with DEFRAG results, both of which are adequate for routine diagnosis. Conclusions We describe one of the largest studies evaluating the Ion Proton based NIPS and the first clinical study reporting pregnancy outcome in a large set of patients. We demonstrate that this platform is highly efficient in detecting the three most common trisomies. Our protocol is robust and can be easily implemented in any medical genetics laboratory

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

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

Validation of the multiplex PCR–serial dilutions for each set of probes.

<p>A: FAM probes (chromosome 21), B: VIC probes (reference chromosome).</p

Observed vs. theoretical chromosome ratios for mixtures of 0.5 ng/μL DNA with a trisomy 21 DNA content of 0%, 5%, 10%, 25% and 50%.

<p>The solid black line corresponds to local polynomial smoothing of the curve with 95% confidence intervals (dashed lines), and the solid gray line represents the expected relationship.</p

Box-and-whisker/Beeswarm plots of the distribution of chromosome ratios in the two groups (upper plot), and the ROC curve for the chromosome ratio as a predictor of trisomy 21 (lower plot) for samples exceeding the threshold for the number of positive PCRs.

<p>In the upper plot, the box displays the median [25^th-75^th percentiles] for the distribution; the whiskers indicate the data points no further than 1.5 times the interquartile range from the box; crosses represent the group means; data points are plotted as open circles; n = 17 and 187 sample points for the trisomy 21 and normal group respectively. In the lower plot, the AUROC is given with its 95% confidence interval(AUC: Area Under the Curve).</p

Validation of the multiplex PCR.

<p>Validation of the multiplex PCR.</p