Characterization of molecular bases of Myhre syndrome

Myhre syndrome (MYHRS, MIM 139210) is a rare developmental disorder first described in 1981, for which about 50 cases are currently reported. Clinical features of MHYRS include typical facial gestalt (short palpebral fissures and philtrum, mid-face hypoplasia, prognathism, narrow mouth), thickened skin, joint limitation, muscular pseudohypertrophy, mild-to-moderate intellectual deficiency and hearing loss. Our group (Caputo et al., 2012) and others (Le Goff et al., 2011a) identified SMAD4 (MIM 600993) as the gene mutated in MYHRS using Whole Exome Sequencing approach. Three different de novo missense changes involving Isoleucine 500 (p.Ile500Thr, p.Ile500Val and p.Ile500Met) within the evolutionary conserved MAD homology 2 domain of SMAD4 were detected in 19 patients. SMAD4 plays a pivotal role in signal transduction of the transforming growth factor beta (TGFβ) superfamily cytokines, which exerts an important role from early embryogenesis to adulthood by mediating transcriptional activation of target genes involved in different cellular processes (such as cell division, differentiation, migration, and programmed cell death). SMAD4 has been established as a tumor suppressor gene, since loss-of-function mutations are known to cause two familiar cancer-prone diseases (juvenile polyposis syndrome (JPS, MIM 174900) and Juvenile polyposis/hereditary hemorrhagic telangiectasia (JPHT, MIM 175050), Gallione et al., 2004; Gallione et al., 2010), and occur in different types of carcinomas (pancreas, gastrointestinal tract and skin). The main purpose of this thesis was the investigation of the molecular bases of Myhre syndrome through functional and in silico approaches. Molecular studies and assays on cell cultures were performed in order to characterize MYHRS causative variants. Firstly, different cellular and biochemical assays were performed, in order to assess MYHRS Ile500 mutations functional impact on SMAD4 protein expression, localization, and on cell proliferation. Western blot analysis of MYHRS fibroblasts and HeLa SMAD4-mutated transfected cells, showed an increased endogenous and ectopic expression of the protein, respectively. Immunofluorescence analysis by confocal laser scanning microscopy of MYHRS fibroblasts disclosed an extra-nuclear accumulation of MYHRS-mutated SMAD4 in patient fibroblasts after 2.5 and 5 hours of TGFβ stimulation. Growth curve of MYHRS-mutated fibroblasts, as well as BrdU incorporation assay on MYHRS fibroblasts and 3T3 transfected cells, demonstrated a reduction in the proliferation levels of both patient and SMAD4-mutated overexpressing cells. Moreover, we collected a cohort of cases with clinical features fitting MYHRS. Molecular screening of SMAD4 coding sequence in these patients identified a novel missense change affecting Arginine 496 (p.Arg496Cys) in three cases. In silico structural analysis, performed in collaboration with Tor Vergata University, suggested that conformational changes promoted by replacement of Arg496 impact the stability of the SMAD heterotrimer and/or proper SMAD4 ubiquitination (Caputo et al., 2014). Since the triplet coding for the Arginine at position 496 encompassed a CpG dinucleotide, we preliminarily investigated the methylation status of the cytosine at nucleotide c.1486, through digestion assays of genomic DNA from leukocytes of control subjects with Hpy99I methylation sensitive restriction enzyme which confirmed that c.1486 C>T mutation localized in a methylated CpG dinucleotide. Finally, the investigation of the parental germline origin of MYHRS mutations was performed. Cloning of genomic fragments encompassing SMAD4 causative mutation and intronic polymorphic site for 11 informative MYHRS cases and segregation analysis demonstrated the paternal germline origin of Myhre pathogenic variants in all these patients, in line with the well-known gender bias in the origin of point mutations. In conclusion, our functional data confirm increased expression of Ile500-mutated SMAD4 in MYHRS affected cells and point out a loss of function effect of MYHRS mutations on fibroblasts proliferation, a mechanism activated by TGFβ signaling. Furthermore, the identification of a new mutation causing this syndrome (c.1486 C>T; p.Arg496Cys), which encompass an amino acid close to Ile500 residue, suggests the impairment of ubiquitination and/or transcription complex stability as the probable outcome of this variant, highlighting the need of further investigations on possible different effect of MYHRS mutations on TGFβ signaling and gene transcription. Finally, the exclusive paternal origin of MYHRS variants in our 11 informative patients expand the number of point mutations causing dominant disorders which display a paternal bias in origin, and lead us to speculate on possibly other mechanisms which could produce a positive selection on MYHRS mutations in male testes.