Variable expression of cerebral cavernous malformations in carriers of a premature termination codon in exon 17 of the Krit1 gene

BACKGROUND: Cerebral cavernous malformations (CCM) present as either sporadic or autosomal dominant conditions with incomplete penetrance of symptoms. Differences in genetic and environmental factors might be minimized among first-degree relatives. We therefore studied clinical expression in a family with several affected members. METHODS: We studied a three-generation family with the onset of CCM as a cerebral haemorrhage in the younger (four-year-old) sibling. Identification and enumeration of CCMs were performed in T2-weighted or gradient-echo MRIs of the whole brains. Genetic analysis comprised SCCP, sequencing and restriction polymorphism of the Krit1 gene in the proband and at risk relatives. RESULTS: The phenotypes of cerebral cavernous malformations (CCMs) in carriers of Krit1 mutations were very variable. We identified a novel frameshift mutation caused by a 1902A insertion in exon 17 of the Krit1 gene, which leads to a premature TAA triplet and predicts the truncating phenotype Y634X. A very striking finding was the absence of both clinical symptoms and CCMs in the eldest sibling harbouring the 1902insA. CONCLUSIONS: Patients in this family, harbouring the same mutation, illustrate the very variable clinical and radiological expression of a Krit1 mutation. The early and critical onset in the proband contrasts with minor clinical findings in affected relatives. This consideration is important in genetic counselling

A Mouse Model of Heritable Cerebrovascular Disease

The study of animal models of heritable cerebrovascular diseases can improve our understanding of disease mechanisms, identify candidate genes for related human disorders, and provide experimental models for preclinical trials. Here we describe a spontaneous mouse mutation that results in reproducible, adult-onset, progressive, focal ischemia in the brain. The pathology is not the result of hemorrhage, embolism, or an anatomical abnormality in the cerebral vasculature. The mutation maps as a single site recessive locus to mouse Chromosome 9 at 105 Mb, a region of shared synteny with human chromosome 3q22. The genetic interval, defined by recombination mapping, contains seven protein-coding genes and one processed transcript, none of which are changed in their expression level, splicing, or sequence in affected mice. Targeted resequencing of the entire interval did not reveal any provocative changes; thus, the causative molecular lesion has not been identified

Developmental timing of CCM2 loss influences cerebral cavernous malformations in mice

As revealed in a new model of cerebral cavernous malformations (CCM), the timing of ablation of Ccm genes determines whether or not CCM lesions arise in brain and retina venous beds

Micro-CT imaging reveals<i> Mekk3 </i>heterozygosity prevents cerebral cavernous malformations in <i>Ccm2</i>-deficient mice

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5μm (leading to a minimum feature size of approximately 25μm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases

KRIT1 Regulates the Homeostasis of Intracellular Reactive Oxygen Species

KRIT1 is a gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries that predispose to seizures, focal neurological deficits, and fatal intracerebral hemorrhage. Comprehensive analysis of the KRIT1 gene in CCM patients has suggested that KRIT1 functions need to be severely impaired for pathogenesis. However, the molecular and cellular functions of KRIT1 as well as CCM pathogenesis mechanisms are still research challenges. We found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. In particular, we demonstrate that KRIT1 loss/down-regulation is associated with a significant increase in intracellular ROS levels. Conversely, ROS levels in KRIT1−/− cells are significantly and dose-dependently reduced after restoration of KRIT1 expression. Moreover, we show that the modulation of intracellular ROS levels by KRIT1 loss/restoration is strictly correlated with the modulation of the expression of the antioxidant protein SOD2 as well as of the transcriptional factor FoxO1, a master regulator of cell responses to oxidative stress and a modulator of SOD2 levels. Furthermore, we show that the KRIT1-dependent maintenance of low ROS levels facilitates the downregulation of cyclin D1 expression required for cell transition from proliferative growth to quiescence. Finally, we demonstrate that the enhanced ROS levels in KRIT1−/− cells are associated with an increased cell susceptibility to oxidative DNA damage and a marked induction of the DNA damage sensor and repair gene Gadd45α, as well as with a decline of mitochondrial energy metabolism. Taken together, our results point to a new model where KRIT1 limits the accumulation of intracellular oxidants and prevents oxidative stress-mediated cellular dysfunction and DNA damage by enhancing the cell capacity to scavenge intracellular ROS through an antioxidant pathway involving FoxO1 and SOD2, thus providing novel and useful insights into the understanding of KRIT1 molecular and cellular functions

Etude en immunohistochimie et RT-PCR en temps réel du rôle pronostique de CXCR4/CXCL12 dans le cancer du sein

Le cancer du sein résulte d'altérations moléculaires successives qui transforment les cellules normales en cellules tumorales avec un potentiel invasif local et métastatique. Les cellules tumorales produisent des métastases préférentiellement au niveau d'organes cibles, phénomène auquel participent les chémokines et leurs récepteurs. Des chémokines comme le CXCL12 et son récepteur, le CXCR4 ont été récemment impli qués dans le processus métastatique dans des études in vitro et dans des modèles animaux et constituent ainsi une cible thérapeutique intéressante. Nous avons analysé le niveau d'expression des transcrits de CX('R-1 et CX('L1? sur 113 carcinomes mam maires par RT PCR quantitative ainsi que l'expression de la protéine CXCR4 par immunohistochimie. Dans un premier temps, nous avons tenté de corréler les résultats de l'immunohistochimie à ceux de la biologie moléculaire puis avons confronté les résultats obtenus aux données cliniques et aux facteurs pronostiques déjà identifiés. Nous avons trouvé que la survenue de métastases est corrélée de façon significative au niveau d'expression du transcrit court du CXCR4(p=0.05). Le niveau d'expression du transcrit long du CXCR4 n'est pas corrélé à la survenue de métastases mais pourrait avoir un impact sur la survie sans récidive (p=0.06). Il n'a pas été retrouvé de corrélation significative entre la survenue de métastases et l'expression des transcrits alternatifs du CXCL12, néanmoins les niveaux d'expression sont plus bas dans les tumeurs métastatiques. Le CXCR4 détecté en immunohistochimie n'est pas relié à l'expression des transcrits long et court du CXCR4 et n'apparaît pas comme un facteur de risque de métastase. Malgré le faible nombre d'événements métastatiques, ce travail confirme la pertinence de caractériser le couple CXCR4/CXCL12 dans le processus métastatique, en particulier le rôle des transcrits alternatifs, et l'intérêt de cette voie en tant que cible thérapeutique potentielle.ROUEN-BU Médecine-Pharmacie (765402102) / SudocSudocFranceF

IDENTIFICATION DU GENE CCM1 IMPLIQUE DANS LES CAVERNOMATOSES CEREBRALES FAMILIALES

LES CAVERNOMES CEREBRAUX SONT DES MALFORMATIONS VASCULAIRES DU SYSTEME NERVEUX CENTRAL. LEUR PREVALENCE EST ESTIMEE A 0.1% ; ENVIRON 20% DES CAS CORRESPONDENT A UNE MALADIE HEREDITAIRE A TRANSMISSION AUTOSOMIQUE DOMINANTE. EN 1995, UNE EQUIPE AMERICAINE AVAIT LOCALISE UN PREMIER GENE (CCM1) EN 7Q21. LA STRATEGIE QUE NOUS AVONS CHOISIE POUR AVANCER DANS LA COMPREHENSION DES MECANISMES PHYSIOPATHOLOGIQUES DE CETTE AFFECTION A ETE D'IDENTIFIER CE PREMIER GENE PAR CLONAGE POSITIONNEL. UNE ANALYSE DE LIAISON GENETIQUE CONDUITE SUR 36 FAMILLES NOUS A PERMIS DE CONFIRMER L'HETEROGENEITE GENETIQUE DE CETTE AFFECTION ET D'ESTIMER LA PROPORTION DE FAMILLES FRANCAISES LIEES A CE LOCUS A ENVIRON 65%. LA CARTOGRAPHIE GENETIQUE HAUTE-RESOLUTION NOUS A PERMIS UNE REDUCTION MODESTE DE L'INTERVALLE. UNE APPROCHE BIO-INFORMATIQUE S'APPUYANT SUR LES DONNEES D'UN CONTIG DE CHROMOSOMES ARTIFICIELS DE LEVURES NOUS A PERMIS DE RECONSTITUER 80% DE LA SEQUENCE DE L'INTERVALLE. EN UTILISANT DES METHODES BIOINFORMATIQUES ET MOLECULAIRES, NOUS AVONS ETABLI UNE CARTE TRANSCRIPTIONNELLE DE LA REGION CCM1 ET IDENTIFIE UN TOTAL DE 53 TRANSCRITS POTENTIELS INCLUANT 8 GENES CONNUS CHEZ L'HOMME OU DANS UNE AUTRE ESPECE. LE CRIBLAGE DE 5 DE CES GENES NOUS A PERMIS D'IDENTIFIER CCM1 COMME ETANT LE GENE CODANT POUR LA PROTEINE KRIT1. TOUTES LES MUTATIONS IDENTIFIEES RESULTAIENT EN UNE PROTEINE PUTATIVE TRONQUEE DE SA PARTIE C-TERMINALE, REGION D'INTERACTION AVEC LA PROTEINE RAP1A. LA NATURE DES MUTATIONS OBSERVEES DANS LES FAMILLES POUVAIT EVOQUER SOIT UN MECANISME DE PERTE DE FONCTION, SOIT UN EFFET DOMINANT-NEGATIF. IL EST MAINTENANT NECESSAIRE DE PRECISER LE PROFIL D'EXPRESSION TISSULAIRE DE CETTE PROTEINE KRIT1 DANS LES TISSUS NORMAUX ET PATHOLOGIQUES, SA LOCALISATION SUB-CELLULAIRE ET DE METTRE AU POINT UN MODELE IN VITRO AINSI QU'UN MODELE ANIMAL POUR COMPRENDRE LES EFFETS DES MUTATIONS DECRITES ET MIEUX APPREHENDER LES MECANISMES PHYSIOPATHOLOGIQUES DE CETTE AFFECTION.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF