Rasopathies are a heterogeneous group of disorders that are due to dominant, gain-of-function mutations in genes in the RAS/MAPK pathway. The RAS/MAPK pathway is involved in regulating cell growth, differentiation, and apoptosis. There is marked clinical variation in clinical phenotypes among different rasopathies despite activation of the RAS/MAPK pathway, suggesting that there may be additional biochemical variables depending on the gene mutated. In this thesis, the activation of the end effectors of the RAS/MAPK pathway, MAPK and MEK, were analyzed using Western blots and flow cytometry in NF1, RIT1, and PTPN11 mutated fibroblasts and lymphoblasts. Although the mutated cell lines were expected to have higher levels of activation due to upregulation of the RAS/MAPK pathway, they were found to have decreased activation compared to the control cells. A suggested reason for these results is that negative feedback loops may decrease the level of activation in the pathway by having activated P-MAPK and P-MEK inhibit previous proteins in the pathway.

Mutations in the RAS-RAF-MEK-ERK (MAPK) pathway give rise to a range of developmental disorders collectively referred to as the RASopathies. De novo germline mutations in patients suffering from these syndromes promote similar phenotypes, which include heart abnormalities, characteristic facial features, cutaneous malformations, gastrointestinal malfunctions, failure to thrive and a spectrum of mental retardation. Although many RASopathies patients show a propensity to develop early-onset benign and malignant tumours, Cardio-faciocutaneous (CFC) syndrome patients do not seem to share this predisposition, with the exception of an increased number of naevi. CFC syndrome is caused by mutations in BRAF, MEK1 or MEK2, with the majority of patients harbouring BRAF mutations. Intriguingly, both kinase-activating and kinase-impaired mutations have been identified in CFC patients. Here, I use the zebrafish system to address the activity of the CFC syndrome alleles and the MAPK pathway in a developmental context and test the potential of small molecule inhibitors to restore normal development. I established an assay for the activity of CFC, melanoma and engineered BRAF and MEK human mutated alleles in vivo. Using zebrafish as an animal model organism, a panel of 31 mutant and wild-type BRAF, MEK1 and MEK2 alleles were expressed in early zebrafish embryos to assess their role in development. Irrespective of the predicted kinase activity, all embryos expressing BRAF and MEK mutant alleles reproducibly manifested the cell movement phenotype during gastrulation. Consistent with aberrant fibroblast growth factor (FGF) signalling and defective gastrulation, in situ hybridisation against convergence-extension markers showed misregulated convergence-extension movement patterns in CFC zebrafish embryos. Finally, I performed whole embryo RNA expression microarrays to identify genes regulated downstream of the CFC mutations, and I discuss the potential for a possible link to some of the phenotypes associated with a CFC zebrafish model. I established that the CFC, BRAF and MEK mutant embryos are sensitive to inhibition of MEK signalling by small molecules. Importantly, a time-window of treatment was identified which was sufficient to restore normal gastrulation movements and to prevent the developmental side effects promoted by the inhibitors at later stages of development. In order to begin considering the therapeutic potential of small molecules in developmental disorders (at least in our model system), the effect of low concentrations of the inhibitors in the normal formation of diverse tissues was thoroughly examined during zebrafish development. From these studies, I identified a concentration of MEK inhibitor that could be administered in a continuous fashion to prevent CFC-associated cell movement defects during gastrulation, without additional later developmental defects. Finally, I addressed the role of MEK-ERK signalling in a specific behavioural phenotype in zebrafish. Many RASopathies patients suffer from mental retardation and experience learning and attention difficulties. Research in our laboratory has identified a novel zebrafish behaviour induced by enhanced cAMP signalling, where the zebrafish seek shaded areas in their environment and exhibit frequent defensive shoaling behaviour. I used western blotting to establish that enhanced cAMP signalling activates the MAPK signalling pathway and, in collaboration with members our laboratory, that this phenotype can be suppressed by administration of the PD325901 MEK inhibitor. While we do not yet know the effect of CFC syndrome mutations on this behaviour, we suggest that altered MEK-ERK signalling may underlie important features of vertebrate behaviour.

Indiana University-Purdue University Indianapolis (IUPUI) / Communication is a vital tool used by humans to share information, coordinate behavior, and survive. However, the ability to communicate can become disrupted or remain absent in individuals with neurodevelopmental disorders: two prominent examples include autism spectrum disorders and developmental language disorders, found in nearly 2% and 10% of the population, respectively. Communication disorders are devastating to the autonomy and quality of life of affected individuals, but clinical solutions are limited due to the complex and often unknown neural etiology underlying these conditions. One known disorder with high incidence of disrupted communication is Neurofibromatosis type 1, the genetic disease caused by heterozygosity of the Ras GTPase-activating protein-coding gene NF1. Mice heterozygous for their ortholog of this gene (Nf1+/-) have been shown to recapitulate neuropsychiatric conditions seen in patients. Using a courtship trial paradigm as a model for testing communication, I have demonstrated that Nf1+/- male mice showed deficits in both courtship and non-courtship social behavior as well as a decrease in the number and duration of ultrasonic vocalizations (USVs). Immediate early gene (IEG) immunohistochemistry (IHC) in neurons of courtship-relevant brain regions revealed the Shell of the Nucleus Accumbens (NAcS) as a dysfunctional brain region in Nf1+/- mice compared to WT male mice following courtship trial. Optogenetic targeting of the Nucleus Accumbens (NAc) restored courtship social behaviors and USV number, but not USV duration or non-courtship gestural social behaviors, in Nf1+/- males. This study contributes to a preclinical foundation for understanding etiology of communication disorders in patients.

Les RASopathies avec phénotype Noonan associent le syndrome de Noonan (SN), le syndrome cardio-facio-cutané (SCFC) et le syndrome de Costello (SC). Leurs manifestations dermatologiques restent peu étudiées.Objectifs Colliger les différentes manifestations dermatologiques des SN, SCFC et SC afin d’établir leur nature et leur prévalence, et définir d’éventuelles corrélations phénotype/génotype au sein de chacune de ces affections.Méthodes Les patients atteints d’un SN, SCFC et SC confirmé sur le plan moléculaire par la présence d’une mutation germinale pathogène étaient inclus dans une étude menée durant 5 ans dans les départements de Génétique, Dermatologie et Pédiatrie des CHU de Bordeaux, Marseille, Montpellier, Nancy, Nantes, Paris AP-HP (Hôpital Robert-Debré et Hôpital Necker-Enfants Malades), Rennes, Saint-Pierre (La Réunion) et Toulouse.Résultats Cent quatre-vingt-quatorze patients atteints de SN, SCFC et SC étaient inclus. Cent-vingt et un patients atteints de SN étaient inclus. La tendance aux hématomes était la manifestation la plus fréquente au cours du SN-PTPN11 (53.8%). Les ML et les tâches café-au-lait atypiques (≥3) étaient observés respectivement dans 94% et 80% des SNML liés à des mutations spécifiques de PTPN11. Des formes atypiques de SNML étaient associées au SN-RAF1 et SN-NRAS. En analyse univariée, les patients sans mutation PTPN11 présentaient (i) un risque de troubles de la kératinisation augmenté (P=0.001), dont la kératose pilaire (KP) (P=0.005), l’ulerythema ophryogenes (UO) (P=0.0001) et la kératodermie palmoplantaire (KPP) (P=0.06), et (ii) un risque augmenté d’alopécie du scalp (P=0.035) et des cils (P=0.06) par rapport aux patients sans mutation PTPN11.Quarante-cinq patients atteints de SCFC étaient inclus, parmi lesquels 77,8% avec mutation BRAF. Les anomalies pilaires étaient constantes, incluant une alopécie du scalp et des sourcils et des cheveux frisés ou bouclés respectivement dans 73% et 69% des cas. Une KP, un UO, une KPP et de multiples naevi (MN >50) étaient observés respectivement dans 82%, 44%, 27% et 29% des cas. L’alopécie des sourcils, l’association UO et KPP, la KP diffuse, et les MN constituaient des signes pertinents de différenciation du SCFC avec le SN et le SC. L’acitrétine orale permettait de traiter avec succès la KPP, tandis que le traitement de l’UO par sirolimus à 1% en topique échouait. Aucune corrélation phénotype-génotype n’était notée.Vingt-et-un patients atteints de SC étaient inclus, parmi lesquels 60% avec la mutation p.G12S-HRAS. Les anomalies pilaires étaient constantes, comprenant des cheveux frisés ou bouclés et une alopécie du scalp respectivement dans 70% and 60% des cas. Un acanthosis nigricans, des papillomes, une KPP, et une cutis laxa acrale étaient notés respectivement dans 65%, 65%, 55% et 80% des cas. Des papules linéaires à disposition pavimenteuse de la lèvre supérieure étaient présentes dans 55% des cas, tandis que l’alopécie des sourcils ou le lymphoedème n’étaient pas observés. Aucune corrélation phénotype-génotype n’était notée. Un patient avec SC atypique avec mutation c187_207dup21/p.Glu63_Asp69dup-HRAS présentait un phénotype cutané distinct associant des MN, un lymphoedème et des manifestations dermatologiques de SC, évoquant une nouvelle forme phénotypique de chevauchement entre un SC et un SN et/ou un SCFC.Conclusion La connaissance précise des manifestations dermatologiques des RASopathies et l’identification de thérapeutiques permet une optimisation de leur prise en charge. / Background Data on dermatological manifestations of RASopathies with Noonan phenotype including Noonan syndrome (NS), cardiofaciocutaneous syndrome (CFCS) and Costello syndrome (CS) remain heterogeneous and based on little dermatological expertise.Objectives To describe the dermatological manifestations of NS, CFCS and CS compare them with the literature findings, and test for dermatological phenotype-genotype correlations with or without the presence of PTPN11, BRAF and pG12S-HRAS mutations respectively for NS, CFCS and CS.Methods We performed a large, 5-year, prospective, multicentric, collaborative dermatological and genetic study.Results A total of 194 patients with NS, CFC and CS were included. One hundred and twenty-nine patients with NS were enrolled, including 65 with PTPN11-NS, 34 with PTPN11-NS with multiple lentigines (NSML), and 30 without PTPN11-NS. Easy bruising was the most frequent dermatological finding in PTPN11-NS, present in 53.8%. Multiple lentigines and café-au-lait macules (≥3) were present in, respectively, 94% and 80% of NSML linked to specific mutations of PTPN11. Atypical forms of NSML could be associated with NS with RAF1 or NRAS mutations. In univariate analysis, patients without a PTPN11 mutation showed (i) a significantly higher frequency of keratinization disorders (P=0.001), including keratosis pilaris (P=0.005), ulerythema ophryogenes (P=0.0001) and palmar and/or plantar hyperkeratosis (P=0.06, trend association), and (ii) a significantly higher frequency of scarce scalp hair (P=0.035) and scarce or absent eyelashes (P=0.06, trend association) than those with PTPN11 mutations. Forty-five patients with CFCS were enrolled with mutations in BRAFin 77.8%. Hair abnormalities were ubiquitous, including scarcity or absence of eyebrows and wavy or curly hair respectively in 73% and 69%. Keratosis pilaris (KP), ulerythema ophryogenes (UO), palmo-plantar hyperkeratosis (PPHK), and multiple melanocytic naevi (MMN; MN >50) were noted respectively in 82%, 44%, 27% and 29%. Scarcity or absence of eyebrows, association of UO and PPHK, diffuse KP, and MMN best differentiated CFCS from NS and CS. Oral acitretin may be highly beneficial for therapeutic management of PPHK, whereas treatment of UO by topical 1% sirolimus failed. No significant dermatological phenotype-genotype correlation was determined. Twenty-one patients with CS were enrolled with p.G12S mutation of HRAS identified respectively in 60%. Hair abnormalities were ubiquitous, including wavy or curly hair and scalp alopecia respectively in 70% and 60%. Acanthosis nigricans with pachydermatoglyphia, papillomas with periorificial location, PPHK, and acral cutis laxa were noted respectively in 65%, 65%, 55% and 80%. Cobblestone papillomatous linear papules of the upper lip was present in 55%, while scarcity of eyebrows and lymphedema were almost absent. No significant dermatological phenotype-genotype correlation was determined. One patient with CS with c187_207dup21/p.Glu63_Asp69dup mutation of HRAS had a new distinct cutaneous phenotype including MMN and severe lymphedema similar to NS and/or CFCS findings.Conclusions A thorough knowledge of RASopathies skin manifestations would help in making a positive diagnosis.

Noonan spectrum disorders belong to the RASopathies, a group of clinically related developmental disorders caused by dysregulation of the RAS-MAPK pathway. This thesis describes genetic and clinical investigations of six families with Noonan spectrum disorders. In the first family, the index patient presented with severe Noonan syndrome (NS) and multiple café-au-lait (CAL) spots, while four additional family members displayed multiple CAL spots only. Genetic analysis of four RAS-MAPK genes revealed a de novo PTPN11 mutation and a paternally inherited NF1 mutation, which could explain the atypically severe NS, but not the CAL spots trait in the family. The co-occurrence of two mutations was also present in another patient with a severe/complex NS-like phenotype. Genetic analysis of nine RASopathy-associated genes identified a de novo SHOC2 mutation and a maternally inherited PTPN11 mutation. The latter was also identified in her brother. Both the mother and the brother displayed mild phenotypes of NS. The results from these studies suggest that an additive effect of co-occurring mutations contributes to severe/complex NS phenotypes. The inherent difficulty in diagnosing Noonan spectrum disorders is evident in families with neurofibromatosis-Noonan syndrome (NFNS). An analysis of nine RASopathy-associated genes in a five-generation family with NFNS revealed a novel NF1 mutation in all affected family members. Notably, this family was initially diagnosed with NS and CAL spots. The clinical overlap between NS and NFNS was further demonstrated in three additional NFNS families. An analysis of twelve RASopathy-associated genes revealed three different NF1 mutations, all segregating with the disorder in each family. These mutations have been reported in patients with NF1, but have, to our knowledge, not been associated with NFNS previously. Together, these findings support the notion that NFNS is a variant of NF1. Due to the clinical overlap between NS and NFNS, we propose screening for NF1 mutations in NS patients negative for mutations in NS-associated genes, preferentially when CAL spots are present. In conclusion, this thesis suggests that co-occurrence of mutations or modifying loci in the RAS-MAPK pathway contributes to the clinical variability observed within Noonan spectrum disorders and further demonstrates the importance of accurate genetic diagnosis.

Deregulierung des RAS/MAPK Signalwegs führen nicht nur zur Krebsentstehung, sondern sind auch mitverantwortlich für Entwicklungsstörungen, dieals Keimbahnmutationen in Schlüsselregulatoren des MAPK Signalwegs zurückzuführen sind, werden aufgrund überlappender Phänotypen unter dem Begriff RASopathien subsumiert. Obwohl die Inzidenz für solide Tumore bei diesen Patienten gering ist, wird ein Zusammenhang zum Auftreten verschiedener Leukämieformen deutlich. Im Rahmen dieser Arbeit wurden Mutationen zweier Schlüsselregulatoren des MAPK Signalwegs, PTPN11 und BRAF, hinsichtlich ihrer Fähigkeit zur neoplastischen Transformation analysiert. Zur Durchführung funktioneller Assays wurden Zelllinien mit lentiviraler Vektoren mit NS-, LS- oder NS und Leukämie-assoziierten SHP2 oder CFC-assoz. BRAF Mutationen (Mut), generiert. Die Testung des neoplastischen Potentials erfolgte anhand nicht-tumorigenen humanen sowie an 208F Ratten-Zelllinie. SHP2/BRAF-Mutationen haben eine spindel-ähnliche Zellmorphologie, Proliferation sowie das Dichte- und Anker-unabhängiges Wachstum in 208F gefördert. Diese Ergebnisse sprechen dafür, dass RASopathie-assoziierte Mutationen zu einem Transformationsphänotyp führen können, ähnlich wie die klassischen Ras Onkogenen. Um zu testen, ob Mut-SHP2 das in vivo Tumorwachstum beeinflusst, wurden SHP2-208F-Zellen in Nacktmäuse injiziert. Eine Förderung des Tumorwachstums konnte sowohl durch mut- als auch durch wt-SHP2 beobachtet werden. RASopathie-assoziierte mutierte Proteine führten auch zu einer moderaten Aktivierung des MAPK-Signalwegs. Eine erhöhte Bindungsstärke zu GAB1 konnte mittels ein TAP-Assay ermittelt werden. Auf transkriptioneller Ebene konnte einer Gensignatur, die sowohl durch RASopathien als auch der klassischen onkogenen BRAF identifiziert werden.Die Ergebnisse dieser Studie können für ein besseres Verständnis der Downstream-liegenden Mechanismen von RASopathie-bezogenen Signalwegen und ihrer Beteiligung an der Tumorprogression beitragen / Deregulation of the Ras/MAPK signaling is implicated in a variety of human diseases, including cancer and developmental disorders. The RASopathies are characterized by an overlapping phenotype in patients and result from germline mutations in key regulators of the MAPK signaling cascade. Although the incidence of solid tumors is rather low, reports on different leukemia forms have increased. In this work, a group of mutations in the genes PTPN11 and BRAF were selected for expression in cell lines for a comprehensive molecular and phenotypic characterization. Non-tumorigenic human cell lines and the rat 208F fibroblasts were transduced with lentiviral particles with SHP2/BRAF wildtype (wt), Noonan (NS)-, NS- and leukemia- or LS–associated SHP2 mutations (mut) and CFC-associated BRAF mutations to identify their potential roles in neoplastic transformation. Mutations in both genes promoted cell morphology alterations, cell proliferation, density- and anchorage-independent growth in rat fibroblasts. These results suggested that RASopathies-associated mutations in both genes confer a transformation phenotype in vitro similar to the classical oncogenes. To investigate whether mutations in SHP2 contribute to tumor growth in vivo, 208F cells expressing wt/mut SHP2 were injected in nude mice. Both wt/mut SHP2 expressing cells promoted tumor growth. Additionally, RASopathies-associated mutant SHP2 and BRAF proteins constitutively activate the MAPK signaling in a moderate manner compared to oncogenic BRAF. To identify modifications in the protein interaction of mut-SHP2, TAP assays were performed. Mut-SHP2 proteins showed an increased binding strength to GAB1 compared to wt. Finally, a microarray analysis revealed a gene cluster commonly regulated in both RASopathies and the oncogenic BRAF. The findings of this work might be useful for a better understanding of the downstream mechanisms of RASopathies-related signaling and their involvement in cancer progression.