RASopathies are a family of syndromes affecting development and growth, sharing RAS signaling dysregulation as pathogenetic mechanism. Past work of our group and others have significantly contributed to our understanding of the molecular causes of these diseases. However, a large fraction of RASopathy cases remains unexplained molecularly. Here, I used the nematode C. elegans to reveal novel molecular mechanisms underlying RASopathies, as well as to identify new candidate genes for these group of developmental disorders. C. elegans is an excellent model to study RASopathies since the RAS-MAPK pathway is well conserved in worms, where it plays a crucial role in vulval development. Based on a gene candidacy approach, we identified two germline mutations in RRAS, a gene encoding a small monomeric GTPase controlling cell adhesion, spreading and migration, underlying a rare and atypical form of Noonan syndrome (NS), the most common RASopathy. We also identified somatic RRAS mutations in 2 cases of non-syndromic juvenile myelomonocytic leukaemia (JMML), a childhood myeloproliferative/myelodysplastic disease caused by upregulated RAS signaling. Two of the three identified mutations affected known oncogenic hotspots of RAS genes and conferred variably enhanced RRAS function and stimulus-dependent MAPK activation. Expression of an RRAS mutant homolog in C. elegans enhanced RAS signaling causing a multivulva (Muv) phenotype, and engendered protruding vulva (Pvl), a phenotype previously linked to the RASopathy-causing SHOC2S2G mutant. These findings provided evidence of a functional link between RRAS and MAPK signaling and reveal an unpredicted role of enhanced RRAS function in human disease. Epistatic analyses performed on C. elegans transgenic lines allowed us to establish that the RASopathy-causing SHOC2 and RRAS mutants belong to the same pathway. Within this signaling network, both RAS-1/RRAS and RAS-2/MRAS are downstream to constitutively active SHOC2, with the former being epistatic to the latter. By using a reverse genetic approach based on RNA interference experiments, we demonstrated that the Muv phenotype was completely mediated by LET-60/RAS, while the Pvl phenotype was modulated by the RHO-family small GTPases CDC-42 and RAC1. We then confirmed these results in fibroblasts derived from patients with Mazzanti syndrome (NS with loose anagen hair) and transfected cell lines. In these models, we observed constitutive RAC1 activation and aberrant lamellipodia formation in cells expressing SHOC2S2G compared to wild-type cells. These results suggested RHO GTPases as excellent candidate genes to be mutated in RASopathies. To explore this hypothesis, mutation scanning of RAC1, RAC2 and CDC42 genes was performed in RASopathy patients by targeted resequencing and identified seven different germline CDC42 mutations in 11 unrelated subjects with a variable phenotype partially overlapping NS and predisposing to thrombocytopenia. In vitro biochemical characterization demonstrated a variable impact of the mutations on GTPase activity and defective binding to WASP. In vitro and in vivo (C. elegans) functional characterization of these mutants allowed to define their impact on cell migration and proliferation, as well as on vulval induction and morphogenesis. A first class of mutations was shown to have an hypomorphic effect on processes mediating cell polarized migration, with no effect on the RAS-MAPK signaling, while a second class of mutations had a gain-of-function effect on both cell migration/proliferation and LET-60/RAS-mediated vulval induction. Overall, our data highlighted the possible contribution of dysregulated signaling controlling cell spreading and migration to certain features of RASopathies, such as lymphedema, cardiac defects and lymphocytes infiltration in non-hematopoietic tissues in case of JMML.
C. elegans as a model to identify and functionally characterize novel genes causing RASopathies and other developmental diseases / Pannone, Luca. - (2017 Feb 27).
|Titolo:||C. elegans as a model to identify and functionally characterize novel genes causing RASopathies and other developmental diseases|
|Data di discussione:||27-feb-2017|
|Appartiene alla tipologia:||07a Tesi di Dottorato|