Identification and characterization of new imprinted genes in the human placenta

L’empreinte parentale est un phénomène épigénétique complexe qui régule l’expression mono-allélique de certains gènes en fonction de l’origine parentale de l’allèle. Les gènes soumis à empreinte (GSE) participent au développement placentaire et fœtal. Ces gènes présentent un intérêt du point de vue fondamental notamment pour leur régulation épigénétique, leur rôle dans les échanges fœto-maternels et le développement du système nerveux. Du point de vue clinique, ils sont impliqués dans les pathologies de la grossesse comme la pré-éclampsie et le retard de croissance intra-utérin ainsi que des pathologies neuro-développementales. De nombreux syndromes cliniques associent retard de croissance intra-utérin et déficience intellectuelle suggérant des voies de signalisation communes dans le développement des tissus placentaire et nerveux. La caractérisation de ces GSE pourrait donc permettre la mise en évidence de ces voies. Un peu moins de 100 GSE ont été identifiés à ce jour chez l’Homme. Nous avons donc mené un crible systématique, en utilisant une approche haut débit, pour identifier sans a priori de nouveaux GSE dans le placenta humain. Cette étude pilote a permis dans un premier temps de mettre en évidence sept nouveaux GSE, dont deux présentent, d’après la littérature, une fonction dans le système nerveux : NTM et MAGI2. Dans le présent travail, nous avons étudié 23 nouveaux gènes candidats afin de valider leur statut de GSE, ce qui a conduit à l’identification de trois nouveaux GSE (DSCAM, AIM1 et NCAM2). Nous avons approfondi l’étude pour l’un d’entre eux, à savoir le gène DSCAM qui est localisé sur le chromosome 21, faisant de lui le premier GSE connu sur ce chromosome. DSCAM participe au guidage des neurones lors du développement du système nerveux central et est un gène candidat pour la déficience intellectuelle associée au Down Syndrome. Nous présentons ici les résultats de la caractérisation de ce GSE dans le placenta humain par l’étude (1) des mécanismes épigénétiques, notamment la méthylation des îlots CpG qui pourrait gouverner la mise en place de l’empreinte, (2) de l’expression de DSCAM dans les placentas normaux et pathologiques, ainsi que dans les cellules trophoblastiques humaines avant et après syncytialisation. Par ailleurs, nous présentons les résultats préliminaires d’études dans des modèles animaux et cellulaires. Nous avons évalué le statut d’empreinte dans d’autres espèces (souris et bovin) et d’autres tissus (notamment le cerveau), et étudié l’effet d’un déficit des partenaires moléculaires de DSCAM (souris invalidées pour les gènes Dcc et Netrin-1). Enfin, nous avons utilisé le modèle des cellules endothéliales immortalisées HUVEC pour étudier le rôle de DSCAM dans l’angiogenèse placentaire.Parental imprinting is a complex epigenetic phenomenon that regulates the mono-allelic expression of some genes in a parent-of-origin way. Imprinted genes (IG) contribute to placental and fetal development. These genes are highly interesting for their epigenetic regulation, their role in materno-fetal nutrient exchange and the neuro-development. They are involved in some pregnancy associated diseases such as pre-eclampsia and intrauterine growth retardation as well as in some neuro-developmental imprinting disorders. Many clinical syndromes associate intrauterine growth retardation and intellectual disability suggesting common signaling pathways in the development of placental and nervous tissue. The characterization of IGs could therefore allow their identification. Less than 100 IGs have been identified to date in humans. We carried out a systematic screening, by means of a high-throughput approach, to identify new IGs in the human placenta. This resulted in the identification of seven new IGs, two of which have reported neuronal functions: NTM and MAGI2. In the present work, we investigated 23 new candidate genes in order to validate their imprinting status, which led to the identification of three new IGs (DSCAM, AIM1 and NCAM2). We have further investigated the DSCAM gene, the first one to be identified on chromosome 21. DSCAM participates to the guidance of neurons during central nervous system development and is a candidate gene for intellectual disability in Down Syndrome. We present here a characterization of this IG in the human placenta including (1) the study of epigenetic mechanisms, in particular CpG islands methylation, that could govern the establishment of imprinting in the locus, and (2) the analysis of the expression of DSCAM in normal and pathological placentas, as well as in human trophoblast cells before and after syncytialization. In addition, we present preliminary results from studies in animal and cellular models. We assessed the imprinting status in other species (mouse and bovine) and other tissues (notably the brain), and studied the effect of the invalidation of DSCAM partners (Dcc and Netrin-1 knockout mice). Finally, we used the HUVEC immortalized endothelial cell model to study the role of DSCAM in placental angiogenesis

Identification et caractérisation de nouveaux gènes soumis à l’empreinte parentale dans le placenta humain

Parental imprinting is a complex epigenetic phenomenon that regulates the mono-allelic expression of some genes in a parent-of-origin way. Imprinted genes (IG) contribute to placental and fetal development. These genes are highly interesting for their epigenetic regulation, their role in materno-fetal nutrient exchange and the neuro-development. They are involved in some pregnancy associated diseases such as pre-eclampsia and intrauterine growth retardation as well as in some neuro-developmental imprinting disorders. Many clinical syndromes associate intrauterine growth retardation and intellectual disability suggesting common signaling pathways in the development of placental and nervous tissue. The characterization of IGs could therefore allow their identification. Less than 100 IGs have been identified to date in humans. We carried out a systematic screening, by means of a high-throughput approach, to identify new IGs in the human placenta. This resulted in the identification of seven new IGs, two of which have reported neuronal functions: NTM and MAGI2. In the present work, we investigated 23 new candidate genes in order to validate their imprinting status, which led to the identification of three new IGs (DSCAM, AIM1 and NCAM2). We have further investigated the DSCAM gene, the first one to be identified on chromosome 21. DSCAM participates to the guidance of neurons during central nervous system development and is a candidate gene for intellectual disability in Down Syndrome. We present here a characterization of this IG in the human placenta including (1) the study of epigenetic mechanisms, in particular CpG islands methylation, that could govern the establishment of imprinting in the locus, and (2) the analysis of the expression of DSCAM in normal and pathological placentas, as well as in human trophoblast cells before and after syncytialization. In addition, we present preliminary results from studies in animal and cellular models. We assessed the imprinting status in other species (mouse and bovine) and other tissues (notably the brain), and studied the effect of the invalidation of DSCAM partners (Dcc and Netrin-1 knockout mice). Finally, we used the HUVEC immortalized endothelial cell model to study the role of DSCAM in placental angiogenesis.L’empreinte parentale est un phénomène épigénétique complexe qui régule l’expression mono-allélique de certains gènes en fonction de l’origine parentale de l’allèle. Les gènes soumis à empreinte (GSE) participent au développement placentaire et fœtal. Ces gènes présentent un intérêt du point de vue fondamental notamment pour leur régulation épigénétique, leur rôle dans les échanges fœto-maternels et le développement du système nerveux. Du point de vue clinique, ils sont impliqués dans les pathologies de la grossesse comme la pré-éclampsie et le retard de croissance intra-utérin ainsi que des pathologies neuro-développementales. De nombreux syndromes cliniques associent retard de croissance intra-utérin et déficience intellectuelle suggérant des voies de signalisation communes dans le développement des tissus placentaire et nerveux. La caractérisation de ces GSE pourrait donc permettre la mise en évidence de ces voies. Un peu moins de 100 GSE ont été identifiés à ce jour chez l’Homme. Nous avons donc mené un crible systématique, en utilisant une approche haut débit, pour identifier sans a priori de nouveaux GSE dans le placenta humain. Cette étude pilote a permis dans un premier temps de mettre en évidence sept nouveaux GSE, dont deux présentent, d’après la littérature, une fonction dans le système nerveux : NTM et MAGI2. Dans le présent travail, nous avons étudié 23 nouveaux gènes candidats afin de valider leur statut de GSE, ce qui a conduit à l’identification de trois nouveaux GSE (DSCAM, AIM1 et NCAM2). Nous avons approfondi l’étude pour l’un d’entre eux, à savoir le gène DSCAM qui est localisé sur le chromosome 21, faisant de lui le premier GSE connu sur ce chromosome. DSCAM participe au guidage des neurones lors du développement du système nerveux central et est un gène candidat pour la déficience intellectuelle associée au Down Syndrome. Nous présentons ici les résultats de la caractérisation de ce GSE dans le placenta humain par l’étude (1) des mécanismes épigénétiques, notamment la méthylation des îlots CpG qui pourrait gouverner la mise en place de l’empreinte, (2) de l’expression de DSCAM dans les placentas normaux et pathologiques, ainsi que dans les cellules trophoblastiques humaines avant et après syncytialisation. Par ailleurs, nous présentons les résultats préliminaires d’études dans des modèles animaux et cellulaires. Nous avons évalué le statut d’empreinte dans d’autres espèces (souris et bovin) et d’autres tissus (notamment le cerveau), et étudié l’effet d’un déficit des partenaires moléculaires de DSCAM (souris invalidées pour les gènes Dcc et Netrin-1). Enfin, nous avons utilisé le modèle des cellules endothéliales immortalisées HUVEC pour étudier le rôle de DSCAM dans l’angiogenèse placentaire

Identification et caractérisation de nouveaux gènes soumis à l’empreinte parentale dans le placenta humain

Parental imprinting is a complex epigenetic phenomenon that regulates the mono-allelic expression of some genes in a parent-of-origin way. Imprinted genes (IG) contribute to placental and fetal development. These genes are highly interesting for their epigenetic regulation, their role in materno-fetal nutrient exchange and the neuro-development. They are involved in some pregnancy associated diseases such as pre-eclampsia and intrauterine growth retardation as well as in some neuro-developmental imprinting disorders. Many clinical syndromes associate intrauterine growth retardation and intellectual disability suggesting common signaling pathways in the development of placental and nervous tissue. The characterization of IGs could therefore allow their identification. Less than 100 IGs have been identified to date in humans. We carried out a systematic screening, by means of a high-throughput approach, to identify new IGs in the human placenta. This resulted in the identification of seven new IGs, two of which have reported neuronal functions: NTM and MAGI2. In the present work, we investigated 23 new candidate genes in order to validate their imprinting status, which led to the identification of three new IGs (DSCAM, AIM1 and NCAM2). We have further investigated the DSCAM gene, the first one to be identified on chromosome 21. DSCAM participates to the guidance of neurons during central nervous system development and is a candidate gene for intellectual disability in Down Syndrome. We present here a characterization of this IG in the human placenta including (1) the study of epigenetic mechanisms, in particular CpG islands methylation, that could govern the establishment of imprinting in the locus, and (2) the analysis of the expression of DSCAM in normal and pathological placentas, as well as in human trophoblast cells before and after syncytialization. In addition, we present preliminary results from studies in animal and cellular models. We assessed the imprinting status in other species (mouse and bovine) and other tissues (notably the brain), and studied the effect of the invalidation of DSCAM partners (Dcc and Netrin-1 knockout mice). Finally, we used the HUVEC immortalized endothelial cell model to study the role of DSCAM in placental angiogenesis.L’empreinte parentale est un phénomène épigénétique complexe qui régule l’expression mono-allélique de certains gènes en fonction de l’origine parentale de l’allèle. Les gènes soumis à empreinte (GSE) participent au développement placentaire et fœtal. Ces gènes présentent un intérêt du point de vue fondamental notamment pour leur régulation épigénétique, leur rôle dans les échanges fœto-maternels et le développement du système nerveux. Du point de vue clinique, ils sont impliqués dans les pathologies de la grossesse comme la pré-éclampsie et le retard de croissance intra-utérin ainsi que des pathologies neuro-développementales. De nombreux syndromes cliniques associent retard de croissance intra-utérin et déficience intellectuelle suggérant des voies de signalisation communes dans le développement des tissus placentaire et nerveux. La caractérisation de ces GSE pourrait donc permettre la mise en évidence de ces voies. Un peu moins de 100 GSE ont été identifiés à ce jour chez l’Homme. Nous avons donc mené un crible systématique, en utilisant une approche haut débit, pour identifier sans a priori de nouveaux GSE dans le placenta humain. Cette étude pilote a permis dans un premier temps de mettre en évidence sept nouveaux GSE, dont deux présentent, d’après la littérature, une fonction dans le système nerveux : NTM et MAGI2. Dans le présent travail, nous avons étudié 23 nouveaux gènes candidats afin de valider leur statut de GSE, ce qui a conduit à l’identification de trois nouveaux GSE (DSCAM, AIM1 et NCAM2). Nous avons approfondi l’étude pour l’un d’entre eux, à savoir le gène DSCAM qui est localisé sur le chromosome 21, faisant de lui le premier GSE connu sur ce chromosome. DSCAM participe au guidage des neurones lors du développement du système nerveux central et est un gène candidat pour la déficience intellectuelle associée au Down Syndrome. Nous présentons ici les résultats de la caractérisation de ce GSE dans le placenta humain par l’étude (1) des mécanismes épigénétiques, notamment la méthylation des îlots CpG qui pourrait gouverner la mise en place de l’empreinte, (2) de l’expression de DSCAM dans les placentas normaux et pathologiques, ainsi que dans les cellules trophoblastiques humaines avant et après syncytialisation. Par ailleurs, nous présentons les résultats préliminaires d’études dans des modèles animaux et cellulaires. Nous avons évalué le statut d’empreinte dans d’autres espèces (souris et bovin) et d’autres tissus (notamment le cerveau), et étudié l’effet d’un déficit des partenaires moléculaires de DSCAM (souris invalidées pour les gènes Dcc et Netrin-1). Enfin, nous avons utilisé le modèle des cellules endothéliales immortalisées HUVEC pour étudier le rôle de DSCAM dans l’angiogenèse placentaire

Efficacité de l'IMSI dans une population infertile présentant soit un facteur masculin sévère, soit plusieurs échecs d'implantation embryonnaire après ICSI

PARIS-BIUP (751062107) / SudocSudocFranceF

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

Could sperm grade under high magnification condition predict IMSI clinical outcome?

Objective: The aim of this study was to examine whether injection of first-best morphology grade selected spermatozoa improves live birth rate (LBR) compared to intracytoplasmic morphologically selected sperm injection (IMSI) using second-best grade sperm. Study design: In this prospective observational study, 132 patients were enrolled. Inclusion criteria were the presence of severe male factor (normal spermatozoa <10% in fresh ejaculated semen and <10% in selected sperm according to David's classification) associated with <= 2 previous ICSI failure. Results of IMSI performed with either first- or second-best morphology grade spermatozoa (according to Vanderzwalmen's classification) were compared. IMSI attempts performed using mixed first- and second-best grade spermatozoa were excluded (n = 41). The primary endpoint was LBR. Results: LBR following IMSI was not statistically different using first- (33.3% (13/39)) or second-best morphology grade spermatozoa (28.9% (15/52)). Our study shows that sperm grading under high magnification using Vanderzwalmen's classification is not correlated to IMSI outcome. Conclusion: We do not validate Vanderzwalmen classification in our external and prospective series. These results point out the need for improving our knowledge about the impact of observed vacuoles under high magnification condition. (C) 2014 Elsevier Ireland Ltd. All rights reserved

Next Generation Mapping a novel approach that enables the detection of unbalanced as well as balanced structural variants

International audienceStructural variants (SVs) include large unbalanced (CNVs) and balanced variants (insertions, inversions and translocations). Whereas the detection of unbalanced SVs has been significantly improved by technological breakthrough such as Chromosomal Microarray Analysis (CMA), the detection of balanced SVs still relies on karyotype despite its very low resolution. Massively parallel sequencing enables the detection of some SVs but its use in clinical setting is yet limited by technical and computational challenges, among which the read length. Next Generation Mapping using the Bionano system is a novel non-sequencing based technology. Long high molecular weight DNA fragments are labelled at specific sites and then stretched out into a nano-channel system for fluorescence reading. The labelling pattern is then compared to a reference genome pattern allowing for the identification of SVs without complex bioinformatic analyses. We sought to evaluate the performance of this technology and its ease of use in a routine cytogenetic laboratory. Our study includes 29 patients bearing balanced (11 translocations and 4 inversions) or unbalanced SVs (1 unbalanced translocation, 7 CNVs ranging from 500kb to 4Mb), complex chromosomal rearrangements (n=4), isochromosomes (n=2) and one case of aneuploidy, all previously identified by karyotype or CMA. The results are analysed blindly and then compared to karyotype or CMA results. Preliminary data on four samples show reliable detection of the expected SVs. This approach has the potential to improve the resolution of the pangenome detection of different sorts of SVs, and could hence complement or even replace karyotype and CMA as a unique, simple and comprehensive test. This would have a significant clinical impact for diseases in which balanced SVs are mainly involved, such as reproductive diseases and recurrent miscarriages

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