Identification de variants structuraux et non-codants par approche multiomique chez des patients atteints de troubles du neurodéveloppement

Molecular diagnosis of neurodevelopmental disorders (NDD) relies mainly on exome sequencing, with a diagnostic yield of 31% for isolated NDD and 53% for syndromic NDD. Genome sequencing (GS) has shown better performance; however, its systematic implementation in diagnostics poses new challenges, particularly in the detection and interpretation of structural variants (SV) or variants located in non-coding regions. We have shown that the application of complementary omics techniques, such as clinical RNA-Seq and optical genome mapping, helps improve the diagnostic yield of genome sequencing for complex or difficult-to-interpret cases.Our study confirms that, based on GS alone, some SVs would not have been retained as pathogens/candidates and that it would have been difficult to pose mechanistic hypotheses for complex events. Furthermore, we have identified and reported a deletion of the long non-coding RNA regulator CHASERR, which we associate with a new syndromic NDD. Currently, clinical RNA-Seq and long-fragment sequencing techniques are the subject of intense research. Their application in routine diagnostics has not yet been standardized and will take a long time to improve. Our results shed new light on this gradual introduction in association with GS.Le diagnostic moléculaire des troubles neurodéveloppementaux (TND) repose encore principalement sur le séquençage d’exome, avec un rendement diagnostique plafonnant à 31 % pour les TND isolés et 53 % pour les TND syndromiques. Le séquençage de génome (SG) a montré de meilleures performances mais sa mise en œuvre systématique en diagnostic pose de nouveaux défis, notamment en ce qui concerne la détection et l'interprétation des variants structuraux (SV) ou des variants localisés dans des régions non codantes.Nous avons montré que l'application de techniques omiques complémentaires, telles que le RNA-Seq clinique et la cartographie optique du génome, contribuent à améliorer le rendement diagnostique du séquençage du génome pour des cas complexes ou difficiles à interpréter. Notre étude confirme que, sur la base du SG seul, certains SV n’auraient pas été retenus comme pathogènes/candidats et qu’il aurait été difficile de poser les hypothèses mécanistiques pour les événements complexes. Par ailleurs, nous avons identifié et rapporté une délétion du long ARN non codant régulateur CHASERR que nous associons à un nouveau TND syndromique.Le RNA-Seq clinique et les techniques de séquençage long fragments sont des sujets de recherche en pleine expansion. Leur application en diagnostic de routine n’est pas encore standardisée et nécessitera encore une longue mise au point. Avec nos résultats nous apportons un éclairage nouveau sur cette introduction progressive, en association avec le SG

Neutrophil-derived extracellular vesicles induce endothelial inflammation and damage through the transfer of miRNAs

International audienceThe critical role of neutrophils in pathological inflammation, notably in various autoimmune disorders, is currently the focus of renewed interest. Here, we demonstrate for the first time that activation of neutrophils with various inflammatory stimuli induces the release of extracellular vesicles (EVs) that are internalized by endothelial cells (ECs), thus leading to the transfer of miR-223, miR-142–3p and miR-451 and subsequent endothelial damage. Indeed, while miR-223 has little effect on EC responses, we show that the induced expression of miR-142–3p and miR-451 in ECs results in profound cell damage, especially in inflammatory conditions, characterized by a dramatic increase in cell apoptosis, impaired angiogenic repair responses, and the induction of IL-6, IL-8, CXCL10 and CXCL11 expression. We show that the strong deleterious effect of miR-142–3p may be due in part to its ability to block the activation of ERK1/2 and eNOS-mediated signals in ECs. miR-142–3p also inhibits the expression of RAC1, ROCK2 and CLIC4, three genes that are critical for EC migration and angiogenic responses. Importantly, miR-223, miR-142–3p and miR-451 are markedly increased in kidney biopsies from patients with active ANCA-associated vasculitis, a severe autoimmune disease that is prototypical of a neutrophil-induced microvascular damage. Taken together, our results suggest that miR-142–3p and miR-451 released in EVs by activated neutrophils can target EC to trigger an inflammatory cascade and induce direct vascular damage, and that therapeutic strategies based on the inhibition of these miRNAs in ECs will have implications for neutrophil-mediated inflammatory diseases

Integrating RNA-Seq into genome sequencing workflow enhances the analysis of structural variants causing neurodevelopmental disorders

International audienceBackgroundMolecular diagnosis of neurodevelopmental disorders (NDDs) is mainly based on exome sequencing (ES), with a diagnostic yield of 31% for isolated and 53% for syndromic NDD. As sequencing costs decrease, genome sequencing (GS) is gradually replacing ES for genome-wide molecular testing. As many variants detected by GS only are in deep intronic or non-coding regions, the interpretation of their impact may be difficult. Here, we showed that integrating RNA-Seq into the GS workflow can enhance the analysis of the molecular causes of NDD, especially structural variants (SVs), by providing valuable complementary information such as aberrant splicing, aberrant expression and monoallelic expression. MethodsWe performed trio-GS on a cohort of 33 individuals with NDD for whom ES was inconclusive. RNA-Seq on skin fibroblasts was then performed in nine individuals for whom GS was inconclusive and optical genome mapping (OGM) was performed in two individuals with an SV of unknown significance. ResultsWe identified pathogenic or likely pathogenic variants in 16 individuals (48%) and six variants of uncertain significance. RNA-Seq contributed to the interpretation in three individuals, and OGM helped to characterise two SVs. ConclusionOur study confirmed that GS significantly improves the diagnostic performance of NDDs. However, most variants detectable by GS alone are structural or located in non-coding regions, which can pose challenges for interpretation. Integration of RNA-Seq data overcame this limitation by confirming the impact of variants at the transcriptional or regulatory level. This result paves the way for new routinely applicable diagnostic protocols