Partial Seizures as the First Manifestation of Primary Angiitis of the Central Nervous System

A young woman presented with initial epileptic seizures and multiple ‘mass-like’ lesions seen on computed tomography and magnetic resonance imaging. Given this presentation and her past medical history, a cerebral biopsy was performed to rule out malignancy, but showed a necrotizing cerebral vasculitis. We describe this case to reinforce the view that primary central nervous system vasculitis (PCNSV) should be considered in the differential diagnosis of a cerebral mass, even if uncommon

Mutations in TUBG1, DYNC1H1, KIF5C and KIF2A cause malformations of cortical development and microcephaly.

International audienceThe genetic causes of malformations of cortical development (MCD) remain largely unknown. Here we report the discovery of multiple pathogenic missense mutations in TUBG1, DYNC1H1 and KIF2A, as well as a single germline mosaic mutation in KIF5C, in subjects with MCD. We found a frequent recurrence of mutations in DYNC1H1, implying that this gene is a major locus for unexplained MCD. We further show that the mutations in KIF5C, KIF2A and DYNC1H1 affect ATP hydrolysis, productive protein folding and microtubule binding, respectively. In addition, we show that suppression of mouse Tubg1 expression in vivo interferes with proper neuronal migration, whereas expression of altered γ-tubulin proteins in Saccharomyces cerevisiae disrupts normal microtubule behavior. Our data reinforce the importance of centrosomal and microtubule-related proteins in cortical development and strongly suggest that microtubule-dependent mitotic and postmitotic processes are major contributors to the pathogenesis of MCD

Intramedullary Astrocytomas: Clinico-pathological, Molecular and Epigenetic Specificities

Les astrocytomes intramédullaires (AIMs) sont des tumeurs très rares du système nerveux central (SNC) dont l’incidence est de moins de 1% des tumeurs primitives du SNC. La nouvelle classification selon l’OMS 2016 des gliomes a radicalement changé le diagnostic histologique en intégrant les données moléculaires dans la pratique quotidienne du diagnostic afin de diminuer la subjectivité inhérente au diagnostic morphologique. Il est donc devenu essentiel d'évaluer ces altérations moléculaires afin d'affiner le pronostic du patient et d'orienter la prise en charge clinique. Cependant, la majorité des études sur les astrocytomes du SNC se concentrent sur les tumeurs cérébrales et peu d'études ont été réalisées, à ce jour, sur les AIMs. Les patients reçoivent généralement des modalités de traitement qui ont été établies pour les tumeurs intracérébrales et aucune guideline n'est disponible concernant l'intégration des altérations moléculaires dans le diagnostic, le pronostic et la gestion clinique des patients porteurs d’AIMs. Récemment, le Centre allemand de recherche sur le cancer (DKFZ) et l'Université de Heidelberg ont mis au point un modèle de classification des tumeurs du SNC basé sur la méthylation de l'ADN (c'est-à-dire le Heidelberg Brain Tumor classifier). Ce classificateur est apparu comme un outil de sous-classification des tumeurs du SNC qui peut potentiellement aider à la définition de nouvelles entités. Cependant, aucune étude liée à la méthylation de l'ADN n'a encore été réalisée spécifiquement sur les AIMs. La présente étude vise à corréler les données cliniques, radiologiques, moléculaires et épigénétiques des AIMs afin d'améliorer les connaissances actuelles sur le pronostic et les profils moléculaires de ces tumeurs. Notre hypothèse est que les AIMs sont biologiquement, cliniquement et moléculairement différents de leurs homologues cérébraux, présentent des biomarqueurs distincts et nécessitent une prise en charge clinique spécifique. Dans notre première étude, nous avons recueilli les données clinico-radiologiques et effectué un séquençage ciblé de nouvelle génération (NGS) pour 61 AIMs afin d'identifier les fusions KIAA1549-BRAF, les mutations dans 33 gènes couramment impliqués dans les gliomes ainsi que la co-délétion 1p/19q. Nous avons également effectué une multiplex droplet digital PCR étudiant les altérations moléculaires du gène FGFR1. Cent dix-sept astrocytomes cérébraux ont également été analysés à titre de comparaison. Nous avons observé que l'évaluation du caractère infiltrant/bien délimité utilisé comme première étape du diagnostic différentiel dans la classification de l’OMS de 2016 des astrocytomes n'est pas adaptée aux AIMs, car aucune différence significative en termes de caractère bien délimité/infiltrant n'a été rapportée entre les AIMs de grade I et II. Nous avons observé qu'il n'y a pas d'impact pronostique du grading des AIMs de bas-grade en grade I et grade II en opposition au traitement chirurgical. La fusion KIAA1549-BRAF, l'altération moléculaire la plus fréquemment observée dans les AIMs pilocytiques de grade I, a été décrite dans 38% des AIMs pilocytiques de grade I, mais avec des fréquences et des breakpoints différents de ceux observés dans la cohorte des astrocytomes cérébraux. Nous n’avons pas observé d’altérations moléculaires dans 19% des AIMs pilocytiques de grade I. Alors que les mutations IDH sont les altérations moléculaires les plus fréquentes dans les astrocytomes diffus de grade II cérébraux, des mutations non-classiques d’IDH ont été observées dans seulement deux cas d’AIMs diffus de grade II et associées à un pronostic plus mauvais en comparaison à ce qui est classiquement décrit dans le cerveau. De plus, seuls deux cas ont subi une évolution anaplasique, dont un cas d'AIM diffus de grade II IDH-muté. Les AIMs diffus de grade II IDH-wt étaient associés à un bon pronostic et associés à des altérations moléculaires de la voie MAPK (mutation BRAF V600E et altération moléculaire de FGFR1) similaires aux gliomes diffus de type pédiatrique. Cependant, 57 % des AIMs diffus de grade II n'ont pas présenté d'altérations moléculaires. En revanche, tous les AIMs de haut-grade présentaient au moins une altération moléculaire, la plus fréquente étant la mutation H3F3A K27M associée à un pronostic significativement plus mauvais (survie globale et survie sans récidive/progression de la maladie). Dans le but de ne pas seulement analyser les altérations moléculaires hotspots, mais des biomarqueurs plus larges, nous avons décidé de réaliser une analyse épigénétique des AIMs. L’étude du profil de méthylation de l’ADN évalue 850.000 CpGs spécifiques sur l’ensemble du génome et représente par conséquent une stratégie puissante permettant de mieux caractériser le profil moléculaire de ces tumeurs. Parmi les 61 échantillons, 23 ont été analysés d’un point de vue épigénétique. En utilisant le « Heidelberg Brain Tumor classifier », parmi les 16 échantillons contributifs, seuls trois cas, uniquement des AIMs de haut-grade, ont été correctement classés avec le score calibré recommandé (cs ≥ 0.9). La majorité des cas restants, principalement représentés par des AIMs de bas-grade, ont été soit considérés comme des cas "sans correspondance" (cs < 0,3, n=7), soit classés avec un cs faible (allant de 0,32 à 0,53, n=6), comprenant des classifications incohérentes. Afin d’améliorer la précision diagnostique, nous avons utilisé différentes analyses non-supervisées combinant les cas de notre série et les cas sélectionnés de la cohorte de référence d’Heidelberg. Tout d'abord, cette analyse a démontré que presque tous les plus proches voisins de notre série d’AIMs étaient localisés en intramédullaire. Cela a souligné le rôle de la localisation dans la classification des tumeurs cérébrales. Tous les AIMs H3F3A K27M-mutés se sont classés avec la classe de méthylation (MC) de référence gliome diffus de ligne médiane mutée H3K27M (DMG_K27M) alors que AIMs H3F3A K27M-wt étaient associés à la classe de méthylation de référence astrocytomes pilocytiques anaplasiques (ANA_PA). Comme était suggéré par les altérations moléculaires dans notre première étude, et bien que le « Heidelberg Brain Tumor classifier » n'ait pas réussi à classer les AIMs pilocytiques de grade I, les analyses non-supervisées ont identifié une nouvelle classe de méthylation distincte appelé « astrocytome pilocytique de la moëlle » ("PA_SPINE").En conclusion, ce paysage clinique, morphologique et moléculaire des AIMs suggèrent que les algorithmes diagnostiques classiquement utilisés pour les astrocytomes cérébraux doivent être adaptés pour les AIMs. L'évaluation du caractère infiltrant/bien délimité et le grading des AIMs de grade I et II ne semblent pas avoir les mêmes implications que dans le cerveau. La majorité des AIMs de bas-grade sont IDH-wt et ont un profil clinique et moléculaire plus proche de celui des gliomes diffus de type pédiatrique. Presque que tous les AIMs de haut-grade sont inclus dans l’entité DMG K27M de la classification de l’OMS de 2016 caractérisée à la fois par la mutation spécifique (H3F3A K27M) et la localisation de la ligne médiane. Même si notre cohorte ne comprenait que seize cas, nous sommes capables de proposer des hypothèses concernant une classification spécifique de la méthylation de l’ADN des AIMs avec une CM spécifique « PA_SPINE » et une association des AIMs de haut-grade H3F3A K27M-wt avec la CM de référence ANA_PA. Pris ensemble, ces résultats suggèrent fortement qu'une classification spécifique des AIMs doit être implémentée.Intramedullary astrocytomas (IMAs) are very rare central nervous system (CNS) tumors with an incidence of less than 1% of all CNS tumors. The 2016 WHO (World Health Organization) Classification drastically changed the histopathological diagnosis by integrating molecular data into daily diagnostic practice in order to decrease the inherent subjectivity of the morphological diagnosis. It has therefore become essential to assess these molecular alterations in order to refine the patient's prognosis and guide the clinical management. However, the majority of studies on CNS astrocytomas focus on brain tumors and few studies have been performed, to date, on IMAs. Patients usually receive modalities of treatment that established for intracerebral tumors and currently no guidelines are available regarding the use of molecular alterations in the diagnosis, prognosis and in the clinical management of patients with IMAs. Recently, the German Cancer Research Centre (DKFZ) and Heidelberg University developed a DNA methylation-based classification model for CNS tumors (i.e. Heidelberg Brain Tumor classifier). This classifier has emerged as a tool that can be used to subclassify CNS tumors that may potentially help in the definition of new entities. However, no DNA methylation-related studies have yet been performed specifically on IMAs. The present study aims to study the correlation between the clinical, radiological, molecular and epigenetic data of IMAs to improve the current knowledge on the prognosis and the molecular profile of these tumors. Our hypothesis is that IMAs are biologically, clinically and molecularly different from their brain counterparts and need specific clinical management and biomarkers.In the first study, we collected clinico-radiological data and performed targeted Next-Generation Sequencing for 61 IMAs to identify KIAA1549-BRAF fusions, mutations in 33 genes commonly implicated in gliomas and 1p/19q codeletion. We also performed FGFR1 multiplex droplet digital PCR assay. One hundred seventeen brain astrocytomas were also analysed for comparison. We observed that assessing the infiltrative/well-circumscribed pattern of the tumor, which is used as a first differential diagnosis step in the 2016 WHO classification for astrocytomas, is not adapted for IMAs since no significant differences in terms of infiltrative/well-circumscribed pattern were noted between grade I and grade II IMAs.We reported that the prognostic impact of the grading of LG IMAs into grade I and grade II is not associated with different prognosis, in contrast to the surgical treatment. KIAA1549-BRAF fusion, the most frequently observed in grade I pilocytic IMAs, was described in 38% of grade I pilocytic IMAs but with different frequencies and breakpoints than those observed in the brain astrocytoma cohort. We did not observe any pathogenic molecular alterations in 19% of grade I pilocytic IMAs. While IDH mutations are the most frequent mutations in brain grade II diffuse astrocytomas, non-canonical IDH mutations were only observed in two grade II diffuse IMAs and were associated with a worse prognosis than what is classically described in the brain. Moreover, only two cases underwent anaplastic evolution, including one IDH-mutant IMA case. IDH wild-type (wt) grade II diffuse IMAs were associated with a good prognosis and harbored MAPK molecular alterations (i.e. BRAF V600E mutation and FGFR1 molecular alteration) similar to the pediatric-type diffuse gliomas. However, 57% of the grade II diffuse IMAs did not harbor molecular alterations. In contrast, all of the HG IMAs presented at least one molecular alteration, with the most frequent one being the H3F3A K27M mutation which was significantly associated with worse outcomes (overall survival and event-free survival). In order to not only analyze the molecular hotspot alterations but a broader biomarker landscape, we decided to apply epigenetic analysis to IMAs. This DNA methylation profiling assesses the methylation level of 850,000 specific CpGs sites all across the genome and therefore represents a powerful strategy to further characterize the molecular profile of tumors. From 61 cases, 23 IMA samples were therefore epigenetically characterized. Using the Heidelberg Brain Tumor classifier, only three HG IMAs, among the 16 contributive samples, were correctly classified with the recommended calibrated score (cs ≥ 0.9). The majority of the remaining cases, mainly represented by LG IMAs, were either considered as “no-match” cases (cs < 0.3, n=7), or classified with a low cs (ranging from 0.32 to 0.53, n=6), including inconsistent classification. In order to improve diagnostic accuracy, we applied different unsupervised analyses to combine cases from our series and a selected Heidelberg reference cohort. First, this analysis demonstrated that nearly all the nearest neighbors of IMA cases were intramedullary located. This emphasized the major role of location in classifying brain tumors. All H3F3A K27M-mutated IMAs clustered with the diffuse midline glioma, H3K27M-mutant (DMG_K27M) reference methylation class (MC) while H3F3A K27M-wt IMAs were strongly related to anaplastic pilocytic astrocytoma (ANA_PA) reference MC. As suggested by their molecular alterations observed in our first study, and while the Heidelberg Brain Tumor classifier did not succeed in classifying grade I pilocytic IMAs, the unsupervised analysis identified new distinct MC called “spine pilocytic astrocytoma” (“PA_SPINE”).In conclusion, these specific clinico-morphological and molecular landscapes of IMAs suggest that the diagnostic algorithms commonly used for brain astrocytomas must be adapted for IMAs. Assessment of the infiltrative/well-delineated pattern and grading LG in grade I and grade II seem to not have the same implications than in the brain. The majority of LG IMAs are IDH-wt and have a clinical and molecular profile more closely to pediatric-type diffuse gliomas. Nearly all HG IMAs are included in the 2016 WHO classification DMG K27M entity which is characterized by both specific mutation (H3F3A K27M) and midline location. Even though our cohort included only sixteen cases, we are able to propose hypotheses for a specific methylation classification of IMAs with a specific PA_SPINE MC and an association of H3F3A K27M-wt HG IMAs with ANA_PA reference MC. Taken together, these results highly suggest that a specific classification for IMAs has to be implemented.Doctorat en Sciences médicales (Médecine)info:eu-repo/semantics/nonPublishe

Partial seizures as the first manifestation of primary angiitis of the central nervous system

info:eu-repo/semantics/publishe

Initial condition assessment for reaction-diffusion glioma growth models: A translational MRI/histology (in)validation study

Diffuse gliomas are highly infiltrative tumors whose early diagnosis and follow-up usually rely on magnetic resonance imaging (MRI). However, the limited sensitivity of this technique makes it impossible to directly assess the extent of the glioma cell invasion, leading to sub-optimal treatment planing. Reaction-diffusion growth models have been proposed for decades to extrapolate glioma cell infiltration beyond margins visible on MRI and predict its spatial-temporal evolution. These models nevertheless require an initial condition, that is the tumor cell density values at every location of the brain at diagnosis time. Several works have proposed to relate the tumor cell density function to abnormality outlines visible on MRI but the underlying assumptions have never been verified so far. In this work we propose to verify these assumptions by stereotactic histological analysis of a non-operated brain with glioblastoma using a tailored 3D-printed slicer. Cell density maps are computed from histological slides using a deep learning approach. The density maps are then registered to a postmortem MR image and related to an MR-derived geodesic distance map to the tumor core. The relation between the edema outlines visible on T2 FLAIR MRI and the distance to the core is also investigated. Our results suggest that (i) the previously suggested exponential decrease of the tumor cell density with the distance to the tumor core is not unreasonable but (ii) the edema outlines may in general not correspond to a cell density iso-contour and (iii) the commonly adopted tumor cell density value at these outlines is likely overestimated. These findings highlight the limitations of using conventional MRI to derive glioma cell density maps and point out the need of validating other methods to initialize reaction-diffusion growth models and make them usable in clinical practice

GRA12, a Toxoplasma dense granule protein associated with the intravacuolar membranous nanotubular network.

International audienceThe intracellular protozoan parasite Toxoplasma gondii develops within the parasitophorous vacuole (PV), an intracellular niche in which it secretes proteins from secretory organelles named dense granules and rhoptries. Here, we describe a new dense granule protein that should now be referred to as GRA12, and that displays no homology with other proteins. Immunofluorescence and immuno-electron microscopy showed that GRA12 behaves similarly to both GRA2 and GRA6. It is secreted into the PV from the anterior pole of the parasite soon after the beginning of invasion, transits to the posterior invaginated pocket of the parasite where a membranous tubulovesicular network is first assembled, and finally resides throughout the vacuolar space, associated with the mature membranous nanotubular network. GRA12 fails to localise at the parasite posterior end in the absence of GRA2. Within the vacuolar space, like the other GRA proteins, GRA12 exists in both a soluble and a membrane-associated form. Using affinity chromatography experiments, we showed that in both the parasite and the PV soluble fractions, GRA12 is purified with the complex of GRA proteins associated with a tagged version of GRA2 and that this association is lost in the PV membranous fraction

Design and Validation of a Gene-Targeted, Next-Generation Sequencing Panel for Routine Diagnosis in Gliomas.

The updated 2016 World Health Organization (WHO) classification system for gliomas integrates molecular alterations and histology to provide a greater diagnostic and prognostic utility than the previous, histology-based classification. The increasing number of markers that are tested in a correct diagnostic procedure makes gene-targeted, next-generation sequencing (NGS) a powerful tool in routine pathology practice. We designed a 14-gene NGS panel specifically aimed at the diagnosis of glioma, which allows simultaneous detection of mutations and copy number variations, including the 1p/19q-codeletion and Epidermal Growth Factor Receptor (EGFR) amplification. To validate this panel, we used reference mutated DNAs, nontumor and non-glioma samples, and 52 glioma samples that were previously characterized. The panel was then prospectively applied to 91 brain lesions. A specificity of 100% and sensitivity of 99.4% was achieved for mutation detection. Orthogonal methods, such as in situ hybridization and immunohistochemical techniques, were used for validation, which showed high concordance. The molecular alterations that were identified allowed diagnosis according to the updated WHO criteria, and helped in the differential diagnosis of difficult cases. This NGS panel is an accurate and sensitive method, which could replace multiple tests for the same sample. Moreover, it is a rapid and cost-effective approach that can be easily implemented in the routine diagnosis of gliomas.info:eu-repo/semantics/publishe

The electron-bifurcating FeFe-hydrogenase Hnd is involved in ethanol metabolism in Desulfovibrio fructosovorans grown on pyruvate

International audienceDesulfovibrio fructosovorans, a sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of H2 into protons and electrons. Among them, Hnd is an electron-bifurcating hydrogenase, coupling the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2. It was previously hypothesized that its biological function involves the production of NADPH necessary for biosynthetic purposes. However, it was subsequently demonstrated that Hnd is instead a NAD+-reducing enzyme, thus its specific function has yet to be established. To understand the physiological role of Hnd in D. fructosovorans, we compared the hnd deletion mutant with the wild-type strain grown on pyruvate. Growth, metabolites production and comsumption, and gene expression were compared under three different growth conditions. Our results indicate that hnd is strongly regulated at the transcriptional level and that its deletion has a drastic effect on the expression of genes for two enzymes, an aldehyde ferredoxin oxidoreductase and an alcohol dehydrogenase. We demonstrated here that Hnd is involved in ethanol metabolism when bacteria grow fermentatively and proposed that Hnd might oxidize part of the H2 produced during fermentation generating both NADH and reduced ferredoxin for ethanol production via its electron bifurcation mechanism