L'endothéline-1 module le calcium cytosolique et nucléaire ainsi que la prolifération cellulaire et l'apoptose des cellules du muscle lisse aortique humain

Dans cette étude, nous avons premièrement examiné l'effet de l'activation ou l'inhibition des récepteurs de l'ET-1 sur les niveaux de base du [Ca][indice inférieur c] et [Ca][indice inférieur n] dans les cellules du MLVh en culture. Nous avons évalué par la suite la contribution des récepteurs ET[indice inférieur A] et ET[indice inférieur B] vasoconstricteurs dans la réponse calcique soutenue induite par l'ET-1. Ensuite nous avons approfondi nos recherches afin de déteminer l'implication d'une protéine-G sensible au PTX et/ou CTX et l'indépendance de la voie de la protéine kinase C dans la réponse calcique induite par l'ET-1. Dans la deuxième partie des travaux, nous avons caractérisé la localisation et la distribution de l'ET-1 et de ses récepteurs ET[indice inférieur A] et ET[indice inférieur B] dans les cellules du MLVh intactes à l'aide des anticorps spécifiques dirigés contre l'ET-1 et ses récepteurs. Cette partie de l'étude nous a permis de démontrer pour la première fois, que les récepteurs ET[indice inférieur B] et non les récepteurs ET[indice inférieur A] sont présents au niveau nucléaire. Dans la troisième partie de l'étude, nous avons tenté d'élucider le mécanisme d'internalisation des récepteurs ET[indice inférieur A] et ET[indice inférieur B] suite à leur stimulation par l'ET-1 dans les cellules du MLVh. Les travaux réalisés au cours de cette partie d'étude ont montré que les récepteurs ET[indice inférieur A] ainsi que les récepteurs ET[indice inférieur B] sont internalisés en réponse à l'ET-1. Les récepteurs ET[indice inférieur A] , surtout membranaires, se retrouvent localisés au niveau péricnucléaire suite à leur internalisation et sont par la suite recyclés au niveau de la membrane de surface. Finalement, nous avons montré que l'ET-1 n'induit pas une augmentation soutenue de l'expression de la forme phosphorylée des p42/p44 MAPKs. De plus, nous avons montré que la stimulation des récepteurs ET[indice inférieur A] et ET[indice inférieur B] atténue l'apoptose induite par la génistéine dans les cellules quiescentes du muscle lisse vasculaire aortique humain. En conclusion, la présente étude suggère que l'activation des récepteurs ET[indice inférieur A] et ET[indice inférieur B] par l'ET-1 module le calcium cytosolique et nucléaire des cellules du MLVh et induit par la suite leur internalisation et la translocation nucléaire des récepteurs ET[indice inférieur B] avec son ligand. De plus l'activation des récepteurs ET[indice inférieur A] et ET[indice inférieur B] de la sarcolemme n'induit pas l'entrée des cellules du MLVh dans le cycle cellulaire en réponse à l'ET-1 mais par contre semble atténuer l'apoptose induite par la génistéine dans ces cellules."--Résumé abrégé par UMI

Investigating the mechanisms of methotrexate neurotoxicity in patients with childhood leukaemia and long-term survivors.

Background/Objectives Adverse neurological events are common (4-20%) during treatment for pediatric acute lymphoblastic leukaemia (ALL) and include seizures, stroke like syndrome and leukoencephalopathy. In addition, chronic neurotoxicity is emerging as a worrying late effect of treatment with long-term survivors experiencing decreased executive function, processing speed and memory function. Survivors are also at increased risk of experiencing learning difficulties, social withdrawal issues and inattention hyperactivity disorders. Methotrexate, an anti-folate chemotherapy agent, is a mainstay of pediatric leukemia treatment regimens globally and is widely implicated as a cause of these neurological side effects. We hypothesise that methotrexate disrupts DNA methylation via effects on S-adenosyl methionine, a key metabolic component that has previously been described to regulate genes involved in myelination. Design/Methods Using both the oligodendrocytic-like cell line MO3.13 and glial cells derived from induced pluripotent stem cells (iPSC) treated with methotrexate, we assayed for changes in DNA methylation and effects on gene expression using whole-genome methylation arrays and RNAseq, respectively. Genes with corresponding methylation and expression changes were selected for further studies of expression by real-time qPCR and assessment of protein levels. Results We identified DNA methylation and corresponding expression changes in genes involved in neurodevelopmental pathways and neurological disorders. Of particular interest was dose-dependent demethylation and increased gene expression of IRS1, a vital component of insulin signalling pathways that is highly expressed in neural tissue and implicated in regulating cognitive performance. We also detected altered DNA methylation within the PLP1 gene, which encodes the most prevalent protein component of myelin. We found that methotrexate treatment in iPSC-derived oligodendrocytes resulted in increased PLP1 methylation associated with a reduction in PLP1 transcript levels as well as PLP1 protein levels. Conclusions Our work provides insight as to the biological mechanisms behind methotrexate-induced neurological side effects for the first time and implicates altered insulin signalling and myelination pathways as a potential causative factor in neurotoxicity. Further work including the use of animal models is warranted for advancing these results towards informing clinical practice

Artificial intelligence-driven genotype–epigenotype–phenotype approaches to resolve challenges in syndrome diagnostics

Methylation; Splitting; Support vector machineMetilació; Divisió; Màquina de vectors de suportMetilación; División; Máquina de vectores de soporteBackground Decisions to split two or more phenotypic manifestations related to genetic variations within the same gene can be challenging, especially during the early stages of syndrome discovery. Genotype-based diagnostics with artificial intelligence (AI)-driven approaches using next-generation phenotyping (NGP) and DNA methylation (DNAm) can be utilized to expedite syndrome delineation within a single gene. Methods We utilized an expanded cohort of 56 patients (22 previously unpublished individuals) with truncating variants in the MN1 gene and attempted different methods to assess plausible strategies to objectively delineate phenotypic differences between the C-Terminal Truncation (CTT) and N-Terminal Truncation (NTT) groups. This involved transcriptomics analysis on available patient fibroblast samples and AI-assisted approaches, including a new statistical method of GestaltMatcher on facial photos and blood DNAm analysis using a support vector machine (SVM) model. Findings RNA-seq analysis was unable to show a significant difference in transcript expression despite our previous hypothesis that NTT variants would induce nonsense mediated decay. DNAm analysis on nine blood DNA samples revealed an episignature for the CTT group. In parallel, the new statistical method of GestaltMatcher objectively distinguished the CTT and NTT groups with a low requirement for cohort number. Validation of this approach was performed on syndromes with known DNAm signatures of SRCAP, SMARCA2 and ADNP to demonstrate the effectiveness of this approach. Interpretation We demonstrate the potential of using AI-based technologies to leverage genotype, phenotype and epigenetics data in facilitating splitting decisions in diagnosis of syndromes with minimal sample requirement.This work was supported by grants from the Society for the Relief of Disabled Children, Commissioned Paediatric Research at HKCH under The Health and Medical Research Fund (PR-HKU-4), the Agence Nationale de la Recherche “Investissements d’Avenir” program (ANR-10-IAHU-01), MSDAvenir (Devo-Decode project) and AXA (“Tête et Cœur” project). This work was supported by a Simons Foundation Autism Research Initiative (SFARI for RW) and an NSW Genomics Collaborative Grant (to AZ) and the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development (U54HD083091, Genetics Core). Bert Callewaert is a Senior Clinical Investigator of the Research Foundation – Flanders. E.B.O. was supported by the grant from Poznan University of Medical Sciences, Poland ProScience 2022 (502-14-11261860-11962). K.L. is supported by the National Institute for Health and Care Research Doctoral Research Fellowship 302303: The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. NIH P50HD103524 PI Sandra Juul, Genetics Core supported participant enrollment and clinical data collection for UW site. None of the sponsors had any role in the design and conduct of the study; collection, management, analysis and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication

Screening for genes that accelerate the epigenetic aging clock in humans reveals a role for the H3K36 methyltransferase NSD1.

BACKGROUND: Epigenetic clocks are mathematical models that predict the biological age of an individual using DNA methylation data and have emerged in the last few years as the most accurate biomarkers of the aging process. However, little is known about the molecular mechanisms that control the rate of such clocks. Here, we have examined the human epigenetic clock in patients with a variety of developmental disorders, harboring mutations in proteins of the epigenetic machinery. RESULTS: Using the Horvath epigenetic clock, we perform an unbiased screen for epigenetic age acceleration in the blood of these patients. We demonstrate that loss-of-function mutations in the H3K36 histone methyltransferase NSD1, which cause Sotos syndrome, substantially accelerate epigenetic aging. Furthermore, we show that the normal aging process and Sotos syndrome share methylation changes and the genomic context in which they occur. Finally, we found that the Horvath clock CpG sites are characterized by a higher Shannon methylation entropy when compared with the rest of the genome, which is dramatically decreased in Sotos syndrome patients. CONCLUSIONS: These results suggest that the H3K36 methylation machinery is a key component of the epigenetic maintenance system in humans, which controls the rate of epigenetic aging, and this role seems to be conserved in model organisms. Our observations provide novel insights into the mechanisms behind the epigenetic aging clock and we expect will shed light on the different processes that erode the human epigenetic landscape during aging

DNA methylation signature is prognostic of choroid plexus tumor aggressiveness

Abstract: Background: Histological grading of choroid plexus tumors (CPTs) remains the best prognostic tool to distinguish between aggressive choroid plexus carcinoma (CPC) and the more benign choroid plexus papilloma (CPP) or atypical choroid plexus papilloma (aCPP); however, these distinctions can be challenging. Standard treatment of CPC is very aggressive and often leads to severe damage to the young child’s brain. Therefore, it is crucial to distinguish between CPC and less aggressive entities (CPP or aCPP) to avoid unnecessary exposure of the young patient to neurotoxic therapy. To better stratify CPTs, we utilized DNA methylation (DNAm) to identify prognostic epigenetic biomarkers for CPCs. Methods: We obtained DNA methylation profiles of 34 CPTs using the HumanMethylation450 BeadChip from Illumina, and the data was analyzed using the Illumina Genome Studio analysis software. Validation of differentially methylated CpG sites chosen as biomarkers was performed using pyrosequencing analysis on additional 22 CPTs. Sensitivity testing of the CPC DNAm signature was performed on a replication cohort of 61 CPT tumors obtained from Neuropathology, University Hospital Münster, Germany. Results: Generated genome-wide DNAm profiles of CPTs showed significant differences in DNAm between CPCs and the CPPs or aCPPs. The prediction of clinical outcome could be improved by combining the DNAm profile with the mutational status of TP53. CPCs with homozygous TP53 mutations clustered as a group separate from those carrying a heterozygous TP53 mutation or CPCs with wild type TP53 (TP53-wt) and showed the worst survival outcome. Specific DNAm signatures for CPCs revealed AK1, PER2, and PLSCR4 as potential biomarkers for CPC that can be used to improve molecular stratification for diagnosis and treatment. Conclusions: We demonstrate that combining specific DNAm signature for CPCs with histological approaches better differentiate aggressive tumors from those that are not life threatening. These findings have important implications for future prognostic risk prediction in clinical disease management

Truncating SRCAP variants outside the Floating-Harbor syndrome locus cause a distinct neurodevelopmental disorder with a specific DNA methylation signature

Truncating variants in exons 33 and 34 of the SNF2-related CREBBP activator protein (SRCAP) gene cause the neurodevelopmental disorder (NDD) Floating-Harbor syndrome (FLHS), characterized by short stature, speech delay, and facial dysmorphism. Here, we present a cohort of 33 individuals with clinical features distinct from FLHS and truncating (mostly de novo) SRCAP variants either proximal (n = 28) or distal (n = 5) to the FLHS locus. Detailed clinical characterization of the proximal SRCAP individuals identified shared characteristics: developmental delay with or without intellectual disability, behavioral and psychiatric problems, non-specific facial features, musculoskeletal issues, and hypotonia. Because FLHS is known to be associated with a unique set of DNA methylation (DNAm) changes in blood, a DNAm signature, we investigated whether there was a distinct signature associated with our affected individuals. A machine-learning model, based on the FLHS DNAm signature, negatively classified all our tested subjects. Comparing proximal variants with typically developing controls, we identified a DNAm signature distinct from the FLHS signature. Based on the DNAm and clinical data, we refer to the condition as "non-FLHS SRCAP-related NDD.'' All five distal variants classified negatively using the FLHS DNAm model while two classified positively using the proximal model. This suggests divergent pathogenicity of these variants, though clinically the distal group presented with NDD, similar to the proximal SRCAP group. In summary, for SRCAP, there is a clear relationship between variant location, DNAm profile, and clinical phenotype. These results highlight the power of combined epigenetic, molecular, and clinical studies to identify and characterize genotype-epigenotype-phenotype correlations