Analyse de la méthylation d’ADN sur puces : étude comparative d’échantillons de tissus congelés et tissus FFPE

International audienceLes études de méthylome permettent de mesurer sur l’intégralité du génome les niveaux de méthylation des sites CpG qui conditionne l’expression des gènes dans chaque cellules et sont réalisées à partir de tissus cryo-préservés ou fixés. En clinique, la méthode de fixation dans le formol et d’inclusion dans la paraffine (FFPE) représente un moyen incontournable de conservation des biopsies. L’étude du méthylome à partir des ADNs extraits de tissus FFPE,souvent dégradés en raison des conditions de fixation et d’inclusion des tissus, reste un défi. Plusieurs techniques d’analyse de méthylation ont été développées dont la puce Infinium Methylation EPIC (850K) d’Illumina. Cette puce commercialisée depuis 2015, permet d’analyser 850 000 sites de méthylation répartis sur l’ensemble du génome humain. Afin d’améliorer la qualité des ADNs issus de matériel conservé par FFPE et par conséquent la reproductibilité de la méthode, une optimisation de protocole par une étape de « restauration » a été introduite. Dans une étude pilote, nous avons comparé par puce Illumina Infinium Methylation EPIC, le méthylome de tumeurs cérébrales ayant été conservées en parallèle par congélation et en paraffine dans le but d’optimiser l’analyse du méthylome sur tissus FFPE. Nous avons donc comparé des paires de biopsies tumorales : Cryo-préservées versus FFPE. Les résultats montrent une distribution des valeurs β des niveaux de méthylation similaire entre les échantillons FFPE et leurs paires congelées, bien que les intensités des sondes soient plus faibles dans le cas des tissus FFPE dégradés par rapport aux congelés ; elles restent néanmoins exploitables.La corrélation des paires FFPE-congelés reste élevée sur la base des valeurs β des sondes filtrées sur les SNP (r2 moyen = 0.92, intervalle entre 0.86 et 0.98). Une corrélation est aussi observée au niveau des intensités des sondes qui s’hybrident sur le chromosome Y et qui permettent de valider l’appartenance au même sexe des échantillons appariés. Cependant, sur la base des valeurs β de l’ensemble des sondes, la corrélation est faible entre certains échantillons appariés. Cela est probablement lié à la composition cellulaire entre la composante congelée et celle incluse en paraffine. Ces résultats suggèrent que les tissus FFPE peuvent être utilisés, après réalisation du protocole de restauration d’Illumina, pour l’analyse de la méthylation par puces EPIC. Ces analyses ont par ailleurs permis l’identification de différentes sous classes de tumeurs dans les tissus FFPE et congelés et devraient aider à la caractérisation encore en cours de potentiels marqueurs de diagnostic et de pronostic. Cette étude pilote sur tissus FFPE réalisée en collaboration avec le Dr Franck BIELLE (ICM), nous permet de proposer une nouvelle prestation sur notre plateforme, l’analyse de la méthylation sur les échantillons FFPE. Cette nouvelle offre complète le catalogue de puces à ADN de la plateforme P3S

Plasma levels of hsa-miR-152-3p are associated with diabetic nephropathy in patients with type 2 diabetes

International audienceBackground: MicroRNAs (miRNAs) are small non-coding RNAs participating in post-transcriptional regulation of genes. Their key role in modulating the susceptibility to human diseases is now widely recognized, in particular in the context of cardiometabolic disorders. The aim of the present study was to identify miRNAs associated with diabetic nephropathy (DN) in patients with type 2 diabetes (T2D).Methods: A next-generation sequencing-based miRNA profiling was performed in a case-control study for DN in plasma samples of 23 T2D patients with DN (cases) and 23 T2D without (controls). The main associations were confirmed using quantitative reverse transcription-polymerase chain reaction and tested for replication in an independent case-control collection of 100 T2D patients, 50 with DN and 50 without.Results: From the 381 known mature miRNAs that were found highly expressed in the discovery samples, we observed and replicated an association between increased plasma levels of hsa-miR-152-3p and DN (P = 4.03 × 10-4 in the combined samples). Hsa-miR-152-3p plasma levels were further found to be positively correlated (P = 0.003) to plasma osmolarity, a surrogate marker for solute carrier net activity, whose regulation is controlled by several genes including SLC5A3, one of the predicted targets of hsa-miR-152-3p.Conclusions: We observed strong evidence for the association of hsa-miR-152-3p plasma levels and DN in patients with T2D, confirming an association previously observed in patients with type 1 diabetes

Brain tumor with an ATXN1-NUTM1 fusion gene expands the histologic spectrum of NUTM1-rearranged neoplasia

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Increased Fatty Acid Oxidation in Differentiated Proximal Tubular Cells Surviving a Reversible Episode of Acute Kidney Injury

Background/aims: Fatty acid oxidation (FAO), the main source of energy produced by tubular epithelial cells in the kidney, was found to be defective in tubulo-interstitial samples dissected out in kidney biopsies from patients with chronic kidney disease (CKD). Experimental data indicated that this decrease was a strong determinant of renal fibrogenesis, hence a focus for therapeutic interventions. Nevertheless, whether persistently differentiated renal tubules, surviving in a pro-fibrotic environment, also suffer from a decrease in FAO, is currently unknown. Methods: To address this question, we isolated proximal tubules captured ex vivo on the basis of the expression of an intact brush border antigen (Prominin-1) in C57BL6/J mice subjected to a controlled, two-hit model of renal fibrosis (reversible ischemic acute kidney injury (AKI) or sham surgery, followed by angiotensin 2 administration). A transcriptomic high throughput sequencing was performed on total mRNA from these cells, and on whole kidneys. Results: In contrast to mice subjected to sham surgery, mice with a history of AKI displayed histologically more renal fibrosis when exposed to angiotensin 2. High throughput RNA sequencing, principal component analysis and clustering showed marked consistency within experimental groups. As expected, FAO transcripts were decreased in whole fibrotic kidneys. Surprisingly, however, up- rather than down-regulation of metabolic pathways (oxidative phosphorylation, fatty acid metabolism, glycolysis, and PPAR signalling pathway) was a hallmark of the differentiated tubules captured from fibrotic kidneys. Immunofluorescence co-staining analysis confirmed that the expression of FAO enzymes was dependent of tubular trophicity

SHH medulloblastoma in a young adult with a TCF4 germline pathogenic variation

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Hippocampal and neocortical BRAF mutant non‐expansive lesions in focal epilepsies

International audienceAbstract Objective Mesial Temporal Lobe Epilepsy‐associated Hippocampal Sclerosis (MTLE‐HS) is a syndrome associated with various aetiologies. We previously identified CD34‐positive extravascular stellate cells (CD34+ cells) possibly related to BRAF V600E oncogenic variant in a subset of MTLE‐HS. We aimed to identify the BRAF V600E oncogenic variants and characterise the CD34+ cells. Methods We analysed BRAF V600E oncogenic variant by digital droplet Polymerase Chain Reaction in 53 MTLE‐HS samples (25 with CD34+ cells) and nine non‐expansive neocortical lesions resected during epilepsy surgery (five with CD34+ cells). Ex vivo multi‐electrode array recording, immunolabelling, methylation microarray and single nuclei RNAseq were performed on BRAF wildtype MTLE‐HS and BRAF V600E mutant non‐expansive lesion of hippocampus and/or neocortex. Results We identified a BRAF V600E oncogenic variant in five MTLE‐HS samples with CD34+ cells (19%) and in five neocortical samples with CD34+ cells (100%). Single nuclei RNAseq of resected samples revealed two unique clusters of abnormal cells (including CD34+ cells) associated with senescence and oligodendrocyte development in both hippocampal and neocortical BRAF V600E mutant samples. The co‐expression of the oncogene‐induced senescence marker p16 INK4A and the outer subventricular zone radial glia progenitor marker HOPX in CD34+ cells was confirmed by multiplex immunostaining. Pseudotime analysis showed that abnormal cells share a common lineage from progenitors to myelinating oligodendrocytes. Epilepsy surgery led to seizure freedom in eight of the 10 patients with BRAF mutant lesions. Interpretation BRAF V600E underlies a subset of MTLE‐HS and epileptogenic non‐expansive neocortical focal lesions. Detection of the oncogenic variant may help diagnosis and open perspectives for targeted therapies

ETMR-like infantile cerebellar embryonal tumors in the extended morphologic spectrum of DICER1-related tumors

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Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors

Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to co-repressors. We also observed large expansion of CFU-MKs derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34+ cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton Cdc42 and RhoA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34+ cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.status: publishe

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34+ progenitors.

International audienceVariants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34(+) cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34(+) cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels