Jessica Cale

Background: Rare diseases are chronic and debilitating and while individually they affect less than 1 in 2,000 people, collectively they have a huge impact affect around 1.8 million Australians. Approximately 80% of these have a genetic origin, however only 30% of patients receive a formal molecular diagnosis. RASopathies are a group of rare disease that are caused by a mutations in the genes involved in the RAS-MAPK pathway, the most common of which is Noonan syndrome, which is characterised by heart defects, short stature, chest deformities, and specific craniofacial features. This study focuses on the effects of a novel c.173C>T (p.Thr58Ile) mutation, found in the NRAS gene of a patient diagnosed with Noonan-like syndrome, on the localisation and function of the NRAS protein. In doing so this study aimed to validate the role of mutations in the patient’s disease and provide information toward the creation of an experimental pipeline for the validation of other rare variants. Methods: U87-MG cells were transiently transfected with the NRAS constructs tagged with GFP2, and stained with specific antibody markers to determine localisation of the proteins using confocal microscopy. Functional studies included an Annexin V apoptosis assay, using flow cytometry to detect and quantify levels of apoptosis in transfected and untransfected populations, and the prediction of conserved domains using online bioinformatic tools. Results and Conclusions: Preliminary results from this study suggest that there is a difference between the localisation and function of the mutant and wild type proteins. The mutant protein was seen to co-localise with the Golgi apparatus as expected if the mutant protein was constitutively active. However the mutant protein was also observed to co-localise with the markers for the plasma membrane, nucleus and endoplasmic reticulum indicating that the mutation affects more than just the activation of the protein. An increase in apoptosis observed in NRASMUT transfected population, in comparison to both the wild type and untransfected populations, indicates that the mutation is engaging the pro-apoptotic function of NRAS associated with an increased activation of the RAS–RAF–MAPKK–MAPK pathway. Bioinformatic analyses identified that the mutation is location within a number of motifs involved in the binding of GTP and thus the activation and inactivation of NRAS, directing further studies toward the proliferation, activation and interaction of NRAS with effectors.