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Targeted therapies against receptor tyrosine kinases improved the prognosis in some patients and provide real therapeutic hope in the fight against cancer. Although the majority of RTK mutations impact the kinase domain and lead to their constitutive activation, those affecting the MET receptor in lung cancer lead to the skipping of exon 14 (MET ex14) encoding the juxtamembrane domain. The lost domain contains at least three negative regulatory sites. Among them, the phosphorylable residues S985 and Y1003 respectively allow the inhibition of kinase activity and the degradation of MET in response to HGF, the ligand of the receptor. The motif ESVD1002, for its part, is a caspase cleavage site allowing the generation of the p40MET fragment involved in apoptosis. In this context, my thesis objective was to characterize the activation mechanisms of the MET ex14 receptor by determining the involvement of HGF and the relative contribution of these regulatory sites in the transforming capacities of MET ex14 cells. To answer these questions, we first established using CRISPR-Cas9 technology a pulmonary epithelial line presenting an exon 14 jump from the 16HBE line. Through collaborative work with a team in London, we have shown that the MET receptor ex14 is dependent of HGF for the establishment of its full transformative capacities. In fact, only 16HBE ex14 cells induce tumor formation and this tumor development is significantly more frequent and presents a higher volume in transgenic mice expressing human HGF. Furthermore, we show that among 18 MET ex14 patients tested, at least a third of them present co-expression of HGF and MET.However, the molecular mechanisms behind this expression and its functional consequences remain to be determined. Secondly, in order to determine the regulatory sites involved in the transforming potential of MET ex14, we established T47D epithelial cell lines expressing wild type MET version, lacking exon 14 or mutated on each or all of the regulatory sites described. Our results show that in response to HGF, the mutation of the Y1003residue induces a stronger and prolonged activation of downstream signaling pathways associated with greatermigratory capacities similar to the MET receptor ex14. Furthermore, the transcriptional program of MET ex14and Y1003F in response to HGF shows similarities, notably including genes associated with invasion. On the otherhand, the mutation of the cleavage site by the caspases induces the loss of the pro-apoptotic capacities of MET, thus promoting the survival of the MET ex14 cells. As T47D MET cells are not able to induce tumor development in vivo, we have also developed a 16HBE Y1003F line mutated by CRISPR technology. Interestingly, the loss of the Y1003 site alone does not confer the same tumorigenic capacities as the MET ex14 mutation in murine xenografts by allowing the development of few tumors compared to MET ex14 cells. Taken together, these results reveal that the loss of exon 14 leads to a novel mode of activation that is different from other kinase activating MET mutations. We show in particular that the MET ex14 receptor is dependent on HGF for the development of a full transforming potential and that these capacities are the consequence of the combined loss of at least two regulatory mechanisms of the receptor impacting migration but also cell survival. These elements support the use of kinase inhibitors targeting MET and could allow HGF to be considered as a potential biomarker of tumor aggressiveness and / or responses to MET inhibitors., Les thérapies ciblant les récepteurs tyrosine kinase ont contribué à améliorer le pronostic de certains patients et apportent un véritable espoir thérapeutique dans la lutte contre les cancers. Bien que la majorité des mutations des RTK impactent le domaine kinase et conduisent à leur activation constitutive, celles touchant le récepteur MET dans le cancer du poumon conduisent au saut de l’exon 14 (MET ex14) codant le domaine juxtamembranaire. Le domaine perdu comporte au moins trois sites de régulation négative. Parmi eux, les résidus phosphorylables S985 et Y1003 permettent respectivement l’inhibition de l’activité kinase et la dégradation de MET en réponse à l’HGF, le ligand du récepteur. Le motif ESVD1002, quant à lui, est un site de clivage par les caspases permettant la génération du fragment p40MET impliqué dans l’apoptose.Dans ce contexte, mon objectif de thèse a été de mieux caractériser les mécanismes d’activation du récepteur MET ex14 en déterminant l’implication de l’HGF et la contribution relative de ces sites de régulation dans les capacités transformantes des cellules MET ex14. Pour répondre à ces questions, dans un premier temps nous avons établi par technologie CRISPR-Cas9 une lignée épithéliale pulmonaire présentant un saut de l’exon 14 à partir de la lignée 16HBE. Au travers d’un travail collaboratif avec une équipe londonienne, nous avons montré que le récepteur MET ex14 est dépendant de l’HGF pour l’établissement de ses capacités transformantes complètes. En effet, seules les cellules 16HBE ex14 induisent la formation de tumeurs et ce développement tumoral est significativement plus fréquent et de volume supérieur dans des souris transgéniques exprimant l’HGF humain. Par ailleurs, nous montrons que parmi 18patients MET ex14 testés, au moins un tiers d’entre eux présentent une co-expression d’HGF et MET. Néanmoins, les mécanismes moléculaires à l’origine de cette expression ainsi que ses conséquences fonctionnelles restent à déterminer. Dans un second temps, afin de mieux caractériser les sites de régulations impliqués dans le potentiel transformant de MET ex14, nous avons établi des lignées cellulaires épithéliales T47D exprimant des versions de MET sauvage, dépourvu de l’exon 14 ou muté sur chacun ou la totalité des sites de régulation décrits. Nos résultats montrent qu’en réponse à l’HGF, la mutation du résidu Y1003 induit une activation des voies de signalisation en aval plus forte et prolongée associée à des capacités migratoires plus importantes similaires au récepteur MET ex14. Par ailleurs, le programme transcriptionnel de MET ex14 et Y1003F en réponse à l’HGF présente des similitudes en incluant notamment des gènes associés à l’invasion. En revanche, la mutation du site de clivage par les caspases induit la perte des capacités pro-apoptotiques de MET favorisant ainsi la survie des cellules MET ex14. Les cellules T47D MET n’induisant pas le développement de tumeurs in vivo, nous avons développé également une lignée 16HBE mutée Y1003F par technologie CRISPR. De manière intéressante, la perte du siteY1003 seule ne confère pas les mêmes capacités tumorigéniques que la mutation MET ex14 en xénogreffes murines en permettant le développement de peu de tumeurs par rapports aux cellules MET ex14. L’ensemble de ces résultats révèle que la perte de l’exon 14 conduit à un mode d’activation original et différent d’autres mutations de MET activatrices de la kinase. Nous montrons notamment que le récepteur MET ex14 est dépendant de l’HGF pour le développement d’un potentiel transformant complet et que ces capacités sont la conséquence de la perte combinée d’au moins deux mécanismes de régulation du récepteur impactant la migration mais également la survie cellulaire. Ces éléments confortent l’utilisation des inhibiteurs kinase ciblant MET et pourraient permettre d’envisager l’HGF comme un potentiel biomarqueur d’agressivité tumoral et/ou de réponses aux inhibiteurs de MET.

Targeted therapies against receptor tyrosine kinases improved the prognosis in some patients and provide real therapeutic hope in the fight against cancer. Although the majority of RTK mutations impact the kinase domain and lead to their constitutive activation, those affecting the MET receptor in lung cancer lead to the skipping of exon 14 (MET ex14) encoding the juxtamembrane domain. The lost domain contains at least three negative regulatory sites. Among them, the phosphorylable residues S985 and Y1003 respectively allow the inhibition of kinase activity and the degradation of MET in response to HGF, the ligand of the receptor. The motif ESVD1002, for its part, is a caspase cleavage site allowing the generation of the p40MET fragment involved in apoptosis. In this context, my thesis objective was to characterize the activation mechanisms of the MET ex14 receptor by determining the involvement of HGF and the relative contribution of these regulatory sites in the transforming capacities of MET ex14 cells. To answer these questions, we first established using CRISPR-Cas9 technology a pulmonary epithelial line presenting an exon 14 jump from the 16HBE line. Through collaborative work with a team in London, we have shown that the MET receptor ex14 is dependent of HGF for the establishment of its full transformative capacities. In fact, only 16HBE ex14 cells induce tumor formation and this tumor development is significantly more frequent and presents a higher volume in transgenic mice expressing human HGF. Furthermore, we show that among 18 MET ex14 patients tested, at least a third of them present co-expression of HGF and MET.However, the molecular mechanisms behind this expression and its functional consequences remain to be determined. Secondly, in order to determine the regulatory sites involved in the transforming potential of MET ex14, we established T47D epithelial cell lines expressing wild type MET version, lacking exon 14 or mutated on each or all of the regulatory sites described. Our results show that in response to HGF, the mutation of the Y1003residue induces a stronger and prolonged activation of downstream signaling pathways associated with greatermigratory capacities similar to the MET receptor ex14. Furthermore, the transcriptional program of MET ex14and Y1003F in response to HGF shows similarities, notably including genes associated with invasion. On the otherhand, the mutation of the cleavage site by the caspases induces the loss of the pro-apoptotic capacities of MET, thus promoting the survival of the MET ex14 cells. As T47D MET cells are not able to induce tumor development in vivo, we have also developed a 16HBE Y1003F line mutated by CRISPR technology. Interestingly, the loss of the Y1003 site alone does not confer the same tumorigenic capacities as the MET ex14 mutation in murine xenografts by allowing the development of few tumors compared to MET ex14 cells. Taken together, these results reveal that the loss of exon 14 leads to a novel mode of activation that is different from other kinase activating MET mutations. We show in particular that the MET ex14 receptor is dependent on HGF for the development of a full transforming potential and that these capacities are the consequence of the combined loss of at least two regulatory mechanisms of the receptor impacting migration but also cell survival. These elements support the use of kinase inhibitors targeting MET and could allow HGF to be considered as a potential biomarker of tumor aggressiveness and / or responses to MET inhibitors., Les thérapies ciblant les récepteurs tyrosine kinase ont contribué à améliorer le pronostic de certains patients et apportent un véritable espoir thérapeutique dans la lutte contre les cancers. Bien que la majorité des mutations des RTK impactent le domaine kinase et conduisent à leur activation constitutive, celles touchant le récepteur MET dans le cancer du poumon conduisent au saut de l’exon 14 (MET ex14) codant le domaine juxtamembranaire. Le domaine perdu comporte au moins trois sites de régulation négative. Parmi eux, les résidus phosphorylables S985 et Y1003 permettent respectivement l’inhibition de l’activité kinase et la dégradation de MET en réponse à l’HGF, le ligand du récepteur. Le motif ESVD1002, quant à lui, est un site de clivage par les caspases permettant la génération du fragment p40MET impliqué dans l’apoptose.Dans ce contexte, mon objectif de thèse a été de mieux caractériser les mécanismes d’activation du récepteur MET ex14 en déterminant l’implication de l’HGF et la contribution relative de ces sites de régulation dans les capacités transformantes des cellules MET ex14. Pour répondre à ces questions, dans un premier temps nous avons établi par technologie CRISPR-Cas9 une lignée épithéliale pulmonaire présentant un saut de l’exon 14 à partir de la lignée 16HBE. Au travers d’un travail collaboratif avec une équipe londonienne, nous avons montré que le récepteur MET ex14 est dépendant de l’HGF pour l’établissement de ses capacités transformantes complètes. En effet, seules les cellules 16HBE ex14 induisent la formation de tumeurs et ce développement tumoral est significativement plus fréquent et de volume supérieur dans des souris transgéniques exprimant l’HGF humain. Par ailleurs, nous montrons que parmi 18patients MET ex14 testés, au moins un tiers d’entre eux présentent une co-expression d’HGF et MET. Néanmoins, les mécanismes moléculaires à l’origine de cette expression ainsi que ses conséquences fonctionnelles restent à déterminer. Dans un second temps, afin de mieux caractériser les sites de régulations impliqués dans le potentiel transformant de MET ex14, nous avons établi des lignées cellulaires épithéliales T47D exprimant des versions de MET sauvage, dépourvu de l’exon 14 ou muté sur chacun ou la totalité des sites de régulation décrits. Nos résultats montrent qu’en réponse à l’HGF, la mutation du résidu Y1003 induit une activation des voies de signalisation en aval plus forte et prolongée associée à des capacités migratoires plus importantes similaires au récepteur MET ex14. Par ailleurs, le programme transcriptionnel de MET ex14 et Y1003F en réponse à l’HGF présente des similitudes en incluant notamment des gènes associés à l’invasion. En revanche, la mutation du site de clivage par les caspases induit la perte des capacités pro-apoptotiques de MET favorisant ainsi la survie des cellules MET ex14. Les cellules T47D MET n’induisant pas le développement de tumeurs in vivo, nous avons développé également une lignée 16HBE mutée Y1003F par technologie CRISPR. De manière intéressante, la perte du siteY1003 seule ne confère pas les mêmes capacités tumorigéniques que la mutation MET ex14 en xénogreffes murines en permettant le développement de peu de tumeurs par rapports aux cellules MET ex14. L’ensemble de ces résultats révèle que la perte de l’exon 14 conduit à un mode d’activation original et différent d’autres mutations de MET activatrices de la kinase. Nous montrons notamment que le récepteur MET ex14 est dépendant de l’HGF pour le développement d’un potentiel transformant complet et que ces capacités sont la conséquence de la perte combinée d’au moins deux mécanismes de régulation du récepteur impactant la migration mais également la survie cellulaire. Ces éléments confortent l’utilisation des inhibiteurs kinase ciblant MET et pourraient permettre d’envisager l’HGF comme un potentiel biomarqueur d’agressivité tumoral et/ou de réponses aux inhibiteurs de MET.

Cathepsin C (CatC) is a cysteine protease involved in a variety of immune and inflammatory pathways such as activation of cytotoxicity of various immune cells. Homozygous or compound heterozygous variants in the CatC coding gene CTSC cause different conditions that have in common severe periodontitis. Periodontitis may occur as part of Papillon-Lefevre syndrome (PLS; OMIM#245000) or Haim-Munk syndrome (HMS; OMIM#245010), or may present as an isolated finding named aggressive periodontitis (AP1; OMIM#170650). AP1 generally affects young children and results in destruction of the periodontal support of the primary dentition. In the present study we report exome sequencing of a three generation consanguineous Turkish family with a recessive form of early-onset AP1. We identified a novel homozygous missense variant in exon 2 of CTSC (NM_148170, c.G302C, p.Trp101Ser) predicted to disrupt protein structure and to be disease causing. This is the first described CTSC variant specific to the nonsyndromic AP1 form. Given the broad phenotypic spectrum associated with CTSC variants, reporting this novel variant gives new insights on genotype/phenotype correlations and might improve diagnosis of patients with early-onset AP1.

Biology flourished during the XXth century and was profoundly disrupted during the last decade because of the transition to the post-genomic era, the spread of high-throughput biology, and the advent of a relatively new discipline, namely bioinformatics. This latter, which encompasses the collection, organization and analysis of biological data using the computer tool, has quickly become inseparable from the studies related to the genome understanding. The consequences of the different mutations that may affect our genes are responsible for a change in the protein sequence and are likely to affect, for example, the stability of the protein, its intracellular targeting, its maturation, its assembly in a multimeric structure, the essential sites for its enzymatic activity or for the interaction with ligands. Thus, a number of bioinformatic developments have made it possible to set up in silico prediction tools of the structure of a protein that is aiming at predicting the impact of local mutations on the structure of proteins. Throughout our study, we have been interested in exploring, through in silico bioinformatic study, three analytical, prediction and modeling, software, choosing as exemple the G12D mutation that affects the proto-oncogene KRAS found in numerous algerian patients with bronchopulmonary cancers cells (NSCLC). This study allowed us to integrate these bioinformatic tools into our laboratory of developmental biology and LBDD differentiation at the University of Oran 1 Ahmed Benbella, in Algeria. Thus, we have been able to conclude, even if the found mutation is predicted to be tolerated and has no deleterious effect on the entire Ras protein, that the consequence of this missense mutation depends mainly on the position in the protein and the chemical properties of the amino acid involved in the substitution and which shows a strong affinity with the GTP molecule.

International audience; Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the DMD gene that encodes the cytoskeletal protein, dystrophin. Dystrophinopathies are inherited in an X-linked recessive manner. Due to the tremendous size of the gene (2.2 megabases), the DMD locus has a high spontaneous mutation rate, and one third of sporadic cases of DMD are due to a de novo mutation. There are seven tissue-specific promoters in the gene. The skeletal muscular transcript contains 79 exons and encode the full-length protein (427-kDa) located at the inner face of the sarcolemma of muscle fibers. DMD gene mutations are highly heterogeneous. Large rearrangements (deletions or duplications of one or more exons) are most frequently involved while point mutations account for 20 %-30 % of cases. A survey of current strategies of molecular diagnosis is presented here. In particular, the role of muscle biopsy (for dystrophin and RNA analyses) in the diagnosis of dystrophinopathies is discussed. In more than 90 % of cases, the clinical severity is correlated with the impact of the mutations on the reading frame and the expression of the dystrophin (absence or residual amount of mutated protein). Various mechanisms contribute to the exceptions. Besides the clinical interest for the patient, the identification of the mutation allows accurate genetic counseling in the familles, and is a necessary prerequisite for the inclusion of the patient in the genotype-based clinical trials.

SummaryDuchenne muscular dystrophy (DMD) is a hereditary disease caused by mutations that disrupt the dystrophin mRNA reading frame. In some cases, forced exclusion (skipping) of a single exon can restore the reading frame, giving rise to a shorter, but still functional, protein. In this study, we constructed lentiviral vectors expressing antisense oligonucleotides in order to induce an efficient exon skipping and to correct the initial frameshift caused by the DMD deletion of CD133+ stem cells. The intramuscular and intra-arterial delivery of genetically corrected CD133 expressing myogenic progenitors isolated from the blood and muscle of DMD patients results in a significant recovery of muscle morphology, function, and dystrophin expression in scid/mdx mice. These data demonstrate that autologous engrafting of blood or muscle-derived CD133+ cells, previously genetically modified to reexpress a functional dystrophin, represents a promising approach for DMD.

Mutations in the FOXL2 gene have recently been shown to cause the blepharophimosis-ptosis-epicanthus inversus syndrome (BPES), a rare genetic disease (MIM 110100).1 In type I BPES eyelid abnormalities are associated with premature ovarian failure (POF), while in type II BPES only the eyelid malformation is observed.2 FOXL2 is the first human autosomal gene whose dominant mutations have been shown to be involved in POF. The eyelid malformation in both BPES types is inherited as a dominant trait and we have recently argued that ovarian failure in type II BPES is a recessive trait.3 FOXL2 is a single exon gene of 2.7 kb. The predicted protein of 376 amino acids belongs to the large family of forkhead/winged helix transcription factors, containing a characteristic 100 amino acid DNA binding forkhead domain. Many members are known to be involved in vertebrate embryogenesis4 and some have been implicated in inherited developmental human disorders.5 Apart from the forkhead domain, the FOXL2 protein also contains a polyalanine (poly-Ala) tract whose role has not yet been elucidated. Recurrent mutations leading to its expansion cause type II BPES and account for 30% of the deleterious alterations detected in the open reading frame (ORF).6,7 These alleles have been considered as hypomorphic (residual activity) in the context of the ovary.1,6 Northern blot analysis and RNA in situ hybridisation have shown that FOXL2 is expressed in developing mouse eyelids and in adult ovarian follicular cells.1 Here we have performed a comparative sequence analysis of FOXL2 in order to study the evolution of the FOXL2 coding region. We have estimated the synonymous (Ks) and non-synonymous (Ka) substitution rates in the ORF of several species, human, goat, mouse and pufferfish. In addition, to determine the subcellular localisation of the FOXL2 protein and its …