Les enzymes de la famille Ten-Eleven-Translocation (TET) sont des oxygénases dépendantes du 2-oxoglutarate et du Fe (II) capables d’hydroxyler les cytosines méthylées. La conversion en 5-hydroxyméthylcytosines constituerait une étape vers la déméthylation et les protéines TET seraient donc impliquées dans le contrôle épigénétique de la transcription. Des mutations inactivatrices acquises du gène TET2 ont été décrites dans environ 20% des hémopathies myéloïdes humaines. L'analyse de deux modèles murins d’invalidation de Tet2 montre que Tet2 contrôle l'hydroxyméthylation et l’homéostasie du compartiment hématopoïétique. Son inactivation entraine des anomalies pléiotropiques des stades précoces et tardifs de l’hématopoïèse et le développement d'hémopathies myéloïdes. L'étude du statut de TET2 chez une grande série de patients porteurs d'hémopathies lymphoïdes matures retrouve des mutations de ce gène chez 12% des cas de lymphomes T. Celles-ci sont significativement associées aux mutations du gène DNMT3A et sont plus fréquemment observées au sein de deux sous-types, le lymphome T angio-immunoblastique et le lymphome T périphérique non spécifié. L’analyse de populations triées montre la préexistence des mutations de TET2 ou de DNMT3A dans les progéniteurs hématopoïétiques chez certains patients.En conclusion, les mutations de TET2 peuvent survenir dans des progéniteurs précoces, leur conférer un avantage sélectif par rapport aux progéniteurs sauvages et conduire au développement d’une hématopoïèse clonale. Des anomalies génétiques additionnelles semblent nécessaires à la transformation tumorale aussi bien myéloïde que lymphoïde. Ces deux types d’hémopathies pourraient donc se développer à partir d’une même atteinte du compartiment des cellules souches hématopoïétiques. / The Ten-Eleven-Translocation (TET) enzymes belong to a family of oxygenases that are dependent on 2-oxoglutarate and Fe (II) and are able to oxidize methylcytosines. This may represent a step toward DNA demethylation and as such these proteins are involved in the epigenetic control of transcription. Acquired TET2 loss-of-function mutations have been reported in about 20% of human myeloid malignancies.Analysis of two Tet2-deficiency mouse models shows that Tet2 controls hydroxymethylation and homeostasis in the hematopoietic compartment. Tet2 deficiency results in pleiotropic abnormalities of both early and late steps of hematopoiesis and leads to the development of myeloid disorders. The sequencing of TET2 in a large series of patients with mature lymphoproliferations identifies TET2 mutations in 12% of T-cell lymphoma. They are significantly associated with DNMT3A mutations and are more frequently observed in two subtypes, the angioimmunoblastic T-cell lymphoma and the peripheral T-cell lymphoma, not other specified. Analysis of flow-sorted populations shows the presence of TET2 and DNMT3A alterations in hematopoietic progenitors in some patients. In summary, TET2 mutations may affect early progenitors, confer a selective advantage compared with controls progenitors and result in a clonal hematopoiesis. Some additional genetic events are likely required to the myeloid or lymphoid transformation. Both diseases could therefore arise from a common alteration of the hematopoietic stem cell compartment.

Afin de mieux connaître les mécanismes moléculaires impliqués dans le cancer du sein, nous avons entrepris l’étude de la fonction du gène suppresseur de tumeur WWOX comprenant le site fragile FRA16D. Nous avons mis en évidence l’association de WWOX avec des composants de la voie de signalisation intracellulaire Wnt/β-caténine : Dvl-2, BCL9 et BCL9-2, ainsi que, l’histone deacetylase 3 (HDAC3). Nous avons défini, pour la première fois, WWOX en tant que nouvel inhibiteur de la voie Wnt/β-caténine. Nos résultats suggèrent que WWOX agit sur cette voie en séquestrant Dvl-2 dans le cytoplasme et en inhibant les activités transcriptionnelles de BCL9 et BCL9-2. En outre, nous avons démontré que HDAC3 est également capable d’inhiber l’activité transcriptionelle de BCL9-2. HDAC3 agirait en recrutant WWOX sur BCL9-2 et cela indépendamment de son activité déacétylase. L’inhibition de la voie Wnt/β-caténine par WWOX suggère que l’inhibition de l’expression de WWOX, souvent observée dans le cancer du sein, pourrait conduire à la suractivation de cette voie et par conséquent à la stimulation de la progression tumorale. En parallèle de ce travail, nous avons étudié l’implication des nouveaux partenaires moléculaires de WWOX que nous avons trouvé dans la carcinogenèse mammaire. Nous avons mis en évidence une surexpression de BCL9, et non pas de BCL9-2, dans les tumeurs du sein, cette surexpression serait due, au moins en partie à des polyploïdies et des amplifications du gène, suggérant un rôle important de BCL9 dans cette pathologie. / In our attempt to better understand the molecular mecanisms in breast carcinogenesis, we studied the role of the tumor suppressor gene WWOX that encompasses the common fragile site FRD16D. We have identified actors of the Wnt/β-catenin pathway as new interactors of WWOX: Dvl-2, BCL9 and BCL9-2 just as HDAC3. We show, for the first time, that WWOX is an inhibitor of the Wnt/β-catenin pathway. Our results suggest that WWOX binds Dvl-2 in the cytoplasm and inhibits BCL9 and BCL9-2’s transcriptionnal activities. Moreover, we have shown that HDAC3 inhibits as well the transcriptional activity of BCL9-2 by recruiting it on WWOX. We then demonstrated that HDAC3 acts independently of its deacetylase activity. The inhibition of the Wnt/b-catenin pathway by WWOX suggests that the down expression of WWOX, frequently found in breast tumors, could trigger the over activation of the Wnt/β-catenin pathway and therefore a tumor progression. In parallel, we have studied the implication of the newly identified molecular partners of WWOX in the mammary carcinogenesis. We have identified an overexpression of BCL9, but not BCL9-2, in a large serie of invasive breast tumors, this overexpression is due, at least in part to polyploidy and gene amplification, suggesting BCL9 plays an important role in this pathology.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Tumor suppressor genes play a role as a growth regulator and a gatekeeper of a cell. Their inactivation is often detected in malignant tumors. Identification of novel tumor suppressor gene candidates may help to further understand tumorigenesis and aid in the discovery of a new treatment leading toward cure of cancer. This PhD research project aimed to understand functional significance of a novel tumor suppressor gene candidate, myosin IC (MYO1C) and to identify potential interaction(s) of the MYO1C protein with key components of the signaling pathways involving in cancer development. In an experimental rat model for endometrial carcinoma (EC), detailed molecular genetic analysis of a candidate tumor suppressor region located distal to the tumor protein 53 (Tp53) suggested the myosin IC gene (Myo1c) as the best potential target for deletion of the genetic material. The question arising was whether and how MYO1C could function as a tumor suppressor gene. By using qPCR, Western blot or immunohistochemistry analyses, we examined MYO1C protein level in panels of well-stratified human colorectal cancer (CRC) and EC respectively. We found that MYO1C was significantly down-regulated in these cancer materials and that for the EC panel, the observed down-regulation of MYO1C correlated with tumor stage, where tumors at more advanced stages had less expression of MYO1C. In cell transfection experiments, we found that over-expression of MYO1C significantly decreased cell proliferation, and silencing MYO1C with siRNA increased cell viability. Additionally, knockdown of MYO1C impaired the ability of cells to migrate, spread and adhere to the surface. Recent published studies suggested a potential interplay between MYO1C and the phosphoinositide 3-kinase (PI3K)/AKT pathway. To examine this hypothesis, we analyzed the expression and/or activation of components of the PI3K/AKT and RAS/ERK signaling pathways in vivo in CRC samples, and in vitro in cells transfected with the MYO1C gene expression construct or MYO1C-targeted siRNA. To identify other potential pathways/ mechanisms through which MYO1C may exert its tumor suppressor activity, we additionally performed new sets of MYO1C-siRNA knockdown experiments. At different time points post transfection, we performed microarray global gene expression experiments followed by bioinformatics analysis of the data. Altogether, the results suggested an early PI3K/AKT response to altered MYO1C expression. We additionally identified several cancer-related genes/pathways with late response to MYO1C knockdown. All things considered, the identification of MYO1C-expression impact on cell proliferation, migration, and adhesion in combination with its interplay between several cancer-related genes and signaling pathways provide further evidence for the initial hypothesis of a tumor suppressor activity of MYO1C. / Cellular, Molecular and Functional Characterization of the Tumor Suppressor Candidate MYO1C

Beyond its essential roles in embryonic development, the Wnt-mediated signal transduction cascade is critically implicated in homeostasis of adult tissues. In the gastrointestinal epithelium, the threshold of active Wnt signaling is kept in a physiological range by a spectrum of regulatory networks and loops, thereby balancing the opposing processes of cell fate determination, proliferation and stem cell self-renewal. Furthermore, compelling evidence undoubtedly link an aberrant Wnt activity to the onset of bowel cancer. Understanding the principle causes and effects secondary to excessive Wnt signaling can provide valuable insights into the pathology of the malignant transformation of the colorectum. The proposed thesis attempts to focus on novel modes of the Wnt pathway modulation; both general and context-specific nuances of the Wnt level adjustment are thereby delineated. The results are presented in three distinct research publications and one review article. The first study examines the contribution of the distinct post-translational modifications, which the Wnt proteins undergo, to their proper processing, secretion and signaling activity. First, we investigated the sequential order and mutual interdependence of cysteine and serine-linked fatty acylation and N-linked glycosylation of murine...

L’hyperplasie bilatérale macronodulaire des surrénales (HBMS) conduit au développement de nodules corticosurrénaliens bilatéraux entraînant un syndrome de Cushing diagnostiqué souvent au cours de la cinquième décennie. L’étiologie de cette maladie n’était que partiellement connue mais le caractère bilatéral de l’atteinte surrénalienne et l’observation de cas familiaux suggéraient, au début de ce travail, une origine génétique. L’analyse de l’ADN tumoral et leucocytaire d’une série de 33 patients opérés a permis de mettre en évidence chez 25% des patients une perte d’hétérozygotie neutre en nombres de copies (LOH) de tout le bras court du chromosome 16 (16p) au niveau du tissu surrénalien. Un séquençage complet du génome de 5 patients (ADN germinal et tumoral) a permis de mettre en évidence une mutation du gène ARMC5 localisé en 16p pour 4 patients. Le séquençage direct des parties codantes d’ARMC5 à partir de l’ADN somatique et germinal de l’ensemble des patients opérés de la cohorte a montré qu’au total, 55% des patients avaient une mutation d’ARMC5. L’inactivation du gène se fait selon la théorie de Knudson (un événement germinal inactivant le premier allèle associé à un autre événement somatique inactivant le second allèle) ce qui laisse supposer qu’ARMC5 est un gène suppresseur de tumeur. L’analyse de la cohorte d’HBMS de nos collaborateurs américains au National Institute Health (laboratoire de CA. Stratakis, Bethesda, USA) a permis de confirmer que les mutations d’ARMC5 étaient un événement fréquent. Des variants de ce gène sont aussi associés à l’hypertension à rénine basse chez les patients noirs-américains. Afin de déterminer des corrélations génotype-phénotype, notre cohorte initiale a été élargie pour constituer une série consécutive de 98 cas index de patients présentant des formes légères à sévères de la maladie, opérées ou non. Vingt-quatre patients (25%) présentaient une altération d’ARMC5. Par ailleurs, 31 nodules surrénaliens de 19 patients ont été analysés en somatique. Le second événement était une mutation dans 68% des cas et une LOH du locus pour les 32% restant. Chez un même patient, le second événement était différent dans chaque nodule présenté. Les patients mutés avaient un syndrome de Cushing plus sévère cliniquement et biologiquement par rapport aux patients non mutés. La taille de leurs surrénales étaient plus grandes avec un plus grand nombre de nodules. Les patients mutés étaient aussi plus jeunes au diagnostic et plus souvent hypertendus. Ces patients étaient ainsi plus souvent opérés. La fonction de la protéine ARMC5 n’était pas connue lors de son identification comme gène de l’HBMS. In vitro, la surexpression du gène sauvage induit l’apoptose. La surexpression des mutants faux-sens et du mutant p.F700del retrouvés chez les patients entraîne moins d’apoptose qu’ARMC5 sauvage. La protéine ARMC5 contient des domaines Armadillo et BTB, connus pour être impliqués dans l’interaction protéine-protéine. En physiologie, l’ACTH stimule la production d’AMPc et la voie de la protéine kinase A (PKA) est impliquée dans différentes pathologies corticosurrénaliennes. Nous avons pu montrer qu’ARMC5 interagissait avec la sous-unité catalytique alpha de la PKA. L’invalidation d’ARMC5 conduit in vitro à une diminution de l’expression d’enzymes de la stéroïdogénèse, de la production de cortisol et une diminution de l’activité PKA. Ainsi, l’hypothèse pour expliquer les HBMS liées à une inactivation d’ARMC5 est que la perte d’apoptose conduit à une hyperplasie nodulaire du tissu corticosurrénalien et que, même si la production de cortisol est diminuée à l’échelle unicellulaire, l’effet de masse global conduit au total à un hypercortisolisme. Nos travaux ont donc identifié et caractérisé un premier gène causal, ARMC5, fréquemment impliqué dans l’HBMS et associé à des formes plus sévères de la maladie. Cette découverte ouvre des perspectives pour le diagnostic familial et la prise en charge des patients. (...) / Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of adrenal Cushing’s syndrome and bilateral adrenal tumors. We suspected a genetic origin of the disease on the basis of the report of some familial cases and the involvement of both adrenal glands. The aim of this study was to find a genetic cause of non syndromic PBMAH. To look at chromosomal abnormalities, we use single-nucleotide polymorphism (SNP) arrays and microsatellite markers analysis in a first series of 33 patients all operated for PBMAH. We realize whole genome sequencing of 5 patients (blood and tumor DNAs matched). Then we genotyped by Sanger sequencing the gene Armadillo Repeat Containing 5 (ARMC5) in this first series and 66 additional patients. Clinical data were collected to establish genotype-phenotype correlation. In addition, the cohort of patients of our collaborators at the National Institute Health (Dr. Stratakis, Bethesda, USA) was studied. The effects of ARMC5 inactivation and overexpression and the partners of the protein were sought in cell-culture models. The most frequent somatic alteration was a loss of heterozygosity at 16p observed in tumors of 25% of the patients. The gene ARMC5, located at 16p11.2, was the most frequently mutated by whole genome sequencing: a mutation was found in 4/5 patients. 55% of the patients of the first cohort (33 patients treated by adrenalectomy for PBMAH) had ARMC5 alteration. One patient presented with germline microdeletion of the locus identified by SNP array. Every patient had two events: either a mutation or a deletion at the germline level, either a second mutation or a LOH at the somatic level. We showed that the two events were present on different alleles suggesting that ARMC5 is a tumor suppressor gene. In addition, we showed for several patients that the second hit was different in each adrenal nodules of a same patient. This first cohort included only operated patients with serious forms of the disease. The study of the American cohort and the analysis of the total cohort of our lab including non-operated patients and milder forms showed an alteration of ARMC5 in about 25% of the patients. Genotype-phenotype correlation showed that ARMC5 defects are associated with younger age at the diagnosis, higher hypercortisolism, bigger adrenals and higher number of nodules. In addition, a mutation of ARMC5 was shown in a patient with a PBMAH secreting both aldosterone and cortisol. Analysis of a series of patient affected by primary hyperaldosteronism suggested that ARMC5 may be associated with hypertension especially in African-American subjet. Overexpression of ARMC5 leads in vitro to cell apoptosis. We showed that this apoptosis was reduced when transfecting vector harboring missense mutations or single amino-acid deletion found in our cohort. Invalidation of ARMC5 leads to a decreased steroidogenic enzymes expression, cortisol production and reduced protein kinase A (PKA) activity. We showed that ARMC5 interacts with the calaytic subunit alpha of the PKA dissociated from the cAMP-bound regulatory subunits. More than one quarter of sporadic PBMAH patients present a pathogenic germline ARMC5 defect and these index cases present a more severe disease. Systematic genotyping of ARMC5 may help for early diagnosis of PBMAH, familial counseling, and patients’ management. ARMC5 appears to be a new regulator of PKA and might represent a new target for the development of pharmacological agents controlling PKA function and cortisol production.

A expressão do gene RECK é ubíqua em tecidos normais e não detectável nas linhagens celulares tumorais testadas e em fibroblastos transformados por diversos oncogenes. Inicialmente isolado como um gene indutor da reversão fenotípica tumoral→normal em fibroblastos transformados por v-Ki-ras, o gene RECK codifica uma glicoproteína de membrana que suprime a invasão tumoral e metástase através da regulação da metaloprotease de matriz-9. Para entender os mecanismos de inibição da expressão do gene RECK, mediada por oncogenes, isolou-se e caracterizou-se a região 5\'- flanqueadora do gene RECK de camundongo (mRECK). Ensaios de atividade promotora utilizando mutantes de deleção da região 5\' - flanqueadora e o gene repórter luciferase, revelaram que a sequência de 52 pb, imediatamente \"upstream\" ao gene, possui uma atividade promotora que é suprimida pelo produto do oncogene Ha-ras (V12). Esta sequência contém dois sítios de ligação a Sp1 (SplA e SplB), um sítio de ligação a cEBPb e um CAAT box. EMSAs e ensaios de atividade promotora, utilizando mutantes pontuais nestes sítios, revelaram que as proteínas Spl e Sp3 se ligam a ambos os sítios Spl, e que a resposta a Ras é mediada apenas pelo sítio SplB. Análise por Southern blot utilizando uma enzima de restrição sensível à metilação e por Northern blot de mRNA extraído de células tratadas com um agente desmetilante, sugeriram que o mecanismo de metilação de DNA não está envolvido na regulação transcricional do gene RECK. O envolvimento de RECK em diversos sistemas de proliferação, invasão e reversão celular foi analisado através de Northern blot. Os resultados sugerem que a expressão de RECK é regulada por soro na linhagem A3l de fibroblastos normais de camundongo. / The RECK gene is ubiquitously expressed in normal human tissues, but is downregulated both in tumor cell lines and in oncogenically transformed fibroblasts. Initially isolated as a tumor→normal phenotypic reversioninducing gene in v-ki-ras-transformed fibroblast, RECK encodes a membrane-anchored glycoprotein that suppresses tumor invasion and metastasis by regulating the matrix metalloproteinase-9. In order to understand the mechanism of oncogene-mediated suppression of RECK gene expression, we have isolated and characterized the 5\'-flanking region of the mouse RECK gene (mRECK). Deletion mutants constructs of this 5\' -flanking region with the luciferase reporter gene, revealed that the 52 base pairs upstream displays promoter activity which is suppressed by the Ha-ras (V12) oncogene. This region contains two Spl-binding motifs (SplA and SplB), one cEBPb-binding motif, and one CAAT box. Gel shift analysis and reporter gene assays, in combination with site-directed point mutations in these elements, revealed that both Spl sites associate with Spl as well as Sp3 proteins, although Ras- responsiveness seems to be mediated only by the downstream Spl site (SplB). Southern blot analysis using a methylation-sensitive restriction enzyme and Northern blot analysis with mRNA extracted from cells treated with a demethylating agent, indicated that the mechanism of DNA methylation is not involved in the regulation of RECK gene transcription. The role of RECK in several systems of cell proliferation, invasion and reversion, analysed by Northern blot, suggested that RECK expression is cell cycle regulated by serum in normal A3l mouse fibroblast cell line.

O complexo da esclerose tuberosa (TSC) é um transtorno genético, sistêmico, com expressividade variável e herança autossômica dominante. Clinicamente manifesta-se devido ao desenvolvimento de hamartomas e hamártias em diferentes tecidos, principalmente no cérebro, rins, coração, pele e pulmões, podendo causar disfunção do órgão. Mutação em um de dois genes supressores tumorais, TSC1 ou TSC2, são responsáveis pelo TSC. Os genes TSC1 e TSC2 codificam para hamartina e tuberina, respectivamente. Ambas as proteínas interagem fisicamente formando um complexo citosólico que inibe mTOR (mammalian target of rapamycin). Testes moleculares para TSC1 e TSC2 são úteis para auxiliar no diagnóstico de casos clínicos difíceis, em aconselhamento genético e estudos de associação genótipo-fenotípica, além de permitirem a caracterização molecular de mecanismos patogenéticos da formação dos hamartomas e análises funcionais de seus produtos gênicos. Apesar de o diagnóstico de TSC ser basicamente clínico, a partir da revisão de seus critérios em 2012 por um grupo de especialistas, o achado de uma mutação em TSC1 ou TSC2 passou a ser considerado suficiente para o diagnóstico definitivo da doença. O estudo apresentado aqui é parte de um projeto em andamento para estabelecer condições ao desenvolvimento de análise de mutações causadoras de TSC nos genes TSC1 e TSC2. Nosso objetivo neste trabalho foi avaliar por sequenciamento de Sanger o DNA genômico de 28 pacientes brasileiros com diagnóstico definitivo de TSC, procedentes dos estados de São Paulo ou Paraná, tendo como alvo a sequência codificadora do gene TSC1, parte de seus segmentos intrônicos, o promotor basal, bem como o segmento imediatamente a 5\' deste. Sete pacientes (25%) apresentaram mutações de sentido trocado (nonsense) ou com deslocamento da leitura à tradução (frameshift) no gene TSC1. Entre 31 outras variantes de DNA encontradas, 23 são polimorfismos conhecidos e oito apresentaram frequência inferior a 1%, como verificado in silico entre mais de mil sequências de genomas humanos. Entre essas oito variantes de DNA novas ou raras, quatro foram detectadas em pacientes para os quais uma mutação patogênica havia sido identificada e, por isso, foram reclassificadas como polimorfismos. Duas e uma variantes de DNA do mesmo paciente flanqueavam um sítio de ligação em potencial para um fator de transcrição específico, a 5\' do promotor basal de TSC1. Por fim, uma nova variante de DNA na região não codificadora do éxon 2 do gene TSC1 foi predita com potencial para alterar um elemento candidato a acentuador de splicing. Em resumo, como observado em estudos anteriores, descrevemos aqui 25% dos pacientes com TSC apresentando mutações patogênicas na sequência codificadora do gene TSC1. Nossos dados mostraram quatro novas variantes de DNA em regiões potencialmente reguladoras da expressão do gene TSC1, que podem revelar-se como mutações patogênicas e, portanto, necessitam ser testadas experimentalmente / Tuberous sclerosis complex (TSC) is a multisystem disorder, with variable expression and autosomal dominant inheritance. Clinically it is due to hamartia and hamartoma development in different tissues, notably in the brain, kidneys, heart, skin and lungs, causing organ dysfunction. Mutations in either tumor suppressor gene, TSC1 or TSC2, are responsible for TSC. TSC1 and TSC2 genes code for hamartin and tuberin, respectively. Both proteins physically interact forming a cytosolic complex that inhibits the mammalian target of rapamycin (mTOR). TSC1 and TSC2 molecular testing has been useful in diagnosing clinically challenging cases, in genetic counseling and genotype/phenotype association studies, besides evaluation of the molecular basis of hamartoma formation and functional analyses of both gene products. Although TSC diagnosis is basically clinical, since the 2012 specialist panel review the finding of a TSC1 or TSC2 mutation has been considered sufficient for the definite diagnosis of the disease. The results presented here are part of an ongoing project to establish conditions for TSC1 and TSC2 mutation studies. Our first aim is to evaluate by Sanger sequencing TSC1 coding sequence, and an average of 132 base pairs of intronic regions next to exon boundaries from TSC patients, in addition to the gene core promoter. We present preliminary results of a sample of 28 patients with definite TSC diagnosis, from São Paulo and Paraná states. Seven patients (25%) displayed TSC1 nonsense or frameshift mutations. Among 31 other DNA variants identified, 23 were known polymorphisms, and eight had frequencies below 1% as verified in silico among more than a thousand human genomes. Out of eight novel or rare DNA variants, four were detected in patients for whom a pathogenic mutation had been found. Two and one additional DNA point variants from the same patient flanked a putative transcription factor binding site. Finally, a novel DNA variant residing in the TSC1 noncoding exon 2 was predicted to change the sequence potential to behave as a splicing enhancer. In summary, similar to previous studies, we describe 25% of TSC patients with mutations in the TSC1 coding sequence. Differently from other reports, our data disclose four novel DNA variants in TSC1 potentially regulatory regions that are likely to unravel novel pathogenic mutations, and thus need to be experimentally tested

O complexo da esclerose tuberosa (TSC) é um transtorno genético, sistêmico, com expressividade variável e herança autossômica dominante. Clinicamente manifesta-se devido ao desenvolvimento de hamartomas e hamártias em diferentes tecidos, principalmente no cérebro, rins, coração, pele e pulmões, podendo causar disfunção do órgão. Mutação em um de dois genes supressores tumorais, TSC1 ou TSC2, são responsáveis pelo TSC. Os genes TSC1 e TSC2 codificam para hamartina e tuberina, respectivamente. Ambas as proteínas interagem fisicamente formando um complexo citosólico que inibe mTOR (mammalian target of rapamycin). Testes moleculares para TSC1 e TSC2 são úteis para auxiliar no diagnóstico de casos clínicos difíceis, em aconselhamento genético e estudos de associação genótipo-fenotípica, além de permitirem a caracterização molecular de mecanismos patogenéticos da formação dos hamartomas e análises funcionais de seus produtos gênicos. Apesar de o diagnóstico de TSC ser basicamente clínico, a partir da revisão de seus critérios em 2012 por um grupo de especialistas, o achado de uma mutação em TSC1 ou TSC2 passou a ser considerado suficiente para o diagnóstico definitivo da doença. O estudo apresentado aqui é parte de um projeto em andamento para estabelecer condições ao desenvolvimento de análise de mutações causadoras de TSC nos genes TSC1 e TSC2. Nosso objetivo neste trabalho foi avaliar por sequenciamento de Sanger o DNA genômico de 28 pacientes brasileiros com diagnóstico definitivo de TSC, procedentes dos estados de São Paulo ou Paraná, tendo como alvo a sequência codificadora do gene TSC1, parte de seus segmentos intrônicos, o promotor basal, bem como o segmento imediatamente a 5\' deste. Sete pacientes (25%) apresentaram mutações de sentido trocado (nonsense) ou com deslocamento da leitura à tradução (frameshift) no gene TSC1. Entre 31 outras variantes de DNA encontradas, 23 são polimorfismos conhecidos e oito apresentaram frequência inferior a 1%, como verificado in silico entre mais de mil sequências de genomas humanos. Entre essas oito variantes de DNA novas ou raras, quatro foram detectadas em pacientes para os quais uma mutação patogênica havia sido identificada e, por isso, foram reclassificadas como polimorfismos. Duas e uma variantes de DNA do mesmo paciente flanqueavam um sítio de ligação em potencial para um fator de transcrição específico, a 5\' do promotor basal de TSC1. Por fim, uma nova variante de DNA na região não codificadora do éxon 2 do gene TSC1 foi predita com potencial para alterar um elemento candidato a acentuador de splicing. Em resumo, como observado em estudos anteriores, descrevemos aqui 25% dos pacientes com TSC apresentando mutações patogênicas na sequência codificadora do gene TSC1. Nossos dados mostraram quatro novas variantes de DNA em regiões potencialmente reguladoras da expressão do gene TSC1, que podem revelar-se como mutações patogênicas e, portanto, necessitam ser testadas experimentalmente / Tuberous sclerosis complex (TSC) is a multisystem disorder, with variable expression and autosomal dominant inheritance. Clinically it is due to hamartia and hamartoma development in different tissues, notably in the brain, kidneys, heart, skin and lungs, causing organ dysfunction. Mutations in either tumor suppressor gene, TSC1 or TSC2, are responsible for TSC. TSC1 and TSC2 genes code for hamartin and tuberin, respectively. Both proteins physically interact forming a cytosolic complex that inhibits the mammalian target of rapamycin (mTOR). TSC1 and TSC2 molecular testing has been useful in diagnosing clinically challenging cases, in genetic counseling and genotype/phenotype association studies, besides evaluation of the molecular basis of hamartoma formation and functional analyses of both gene products. Although TSC diagnosis is basically clinical, since the 2012 specialist panel review the finding of a TSC1 or TSC2 mutation has been considered sufficient for the definite diagnosis of the disease. The results presented here are part of an ongoing project to establish conditions for TSC1 and TSC2 mutation studies. Our first aim is to evaluate by Sanger sequencing TSC1 coding sequence, and an average of 132 base pairs of intronic regions next to exon boundaries from TSC patients, in addition to the gene core promoter. We present preliminary results of a sample of 28 patients with definite TSC diagnosis, from São Paulo and Paraná states. Seven patients (25%) displayed TSC1 nonsense or frameshift mutations. Among 31 other DNA variants identified, 23 were known polymorphisms, and eight had frequencies below 1% as verified in silico among more than a thousand human genomes. Out of eight novel or rare DNA variants, four were detected in patients for whom a pathogenic mutation had been found. Two and one additional DNA point variants from the same patient flanked a putative transcription factor binding site. Finally, a novel DNA variant residing in the TSC1 noncoding exon 2 was predicted to change the sequence potential to behave as a splicing enhancer. In summary, similar to previous studies, we describe 25% of TSC patients with mutations in the TSC1 coding sequence. Differently from other reports, our data disclose four novel DNA variants in TSC1 potentially regulatory regions that are likely to unravel novel pathogenic mutations, and thus need to be experimentally tested

Les cancers du sein de sous-type luminal B sont associés à un mauvais pronostic. Afin de mieux comprendre la biologie de ce sous-type, nous avons étudié au sein de 188 tumeurs mammaires de différents sous-types, les anomalies du nombre de copies, les méthylations de l'ADN, les profils d'expression génique et les mutations somatiques dans 9 gènes sélectionnés. Un total respectif de 237 et 101 oncogènes et gènes suppresseurs de tumeurs (TSG) candidats présentaient une dérégulation de l'expression en relation avec leur CNA. 88% des TSG potentiels étaient localisés sur le bras chromosomique 6q. 101 oncogènes candidats ont été validés sur une série publique de 5765 cancers du sein, et l'expression de 67 gènes était associée à un mauvais pronostic au sein des tumeurs luminales. 24 gènes présentaient une dérégulation de l'expression en relation avec un haut niveau de méthylation de l'ADN. FOXO3, PIK3CA et TP53 étaient les gènes les plus fréquemment mutés parmi les 9 testés. Dans une méta-analyse de séquençage de nouvelle génération regroupant 875 cancers du sein, les gènes les plus fréquemment mutés dans le sous-type luminal B étaient PIK3CA, TP53 et GATA3. Les nombreuses altérations moléculaires ciblaient des voies de signalisation communes, incluant 3 axes pouvant jouer un rôle majeur dans le sous-type luminal B : la voie TP53 et l'instabilité chromosomique, les voies de signalisation PI3K/AKT/MTOR/FOXO et MAPK/JNK, et les altérations des facteurs de transcription et épigénomiques. En conclusion, nous avons établi un répertoire de gènes candidats dans le sous-type luminal B qui pourraient être impliqués dans le développement et/ou l'hormonorésistance de ce sous-type. / Breast cancers (BCs) of the luminal B subtype have a poor prognosis. To better understand this subtype we studied in 188 BCs of various molecular subtypes, DNA copy number aberrations, DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q. 101 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 was associated with poor survival in luminal tumors. 24 genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, PIK3CA, TP53 and GATA3 were the most frequent mutated genes. Numerous molecular alterations targeted common signalling pathway, included 3 ways wich may play a major in the luminal B subtype: TP53 pathway and chromosomal instability, PI3K/AKT/MTOR/FOXO and MAPK/JNK pathway, and epigenomic and transcription factors alterations. In conclusion, we have reported a repertoire of luminal B candidate genes that may be involved in the development and/or hormone resistance of this subtype.