Functional validation of genetic variants identified by next generation sequencing in malformations of cortical development.

Malformations of cortical development (MCDs) result from a disruption in the complex process of the human brain cortex formation and are highly associated to severe epilepsy, neurodevelopmental delay and motor dysfunction. The advent of next-generation sequencing (NGS) has considerably accelerated the identification of MCDs causing genes, greatly increasing their number and related knowledge. So far, more than 100 genes have been associated with one or more types of MCD. However, despite NGS technologies have considerably increased the potential of identifying the causal mutations in patients with MCD, in some cases NGS results have some limits of interpretation and functional studies are needed to clarify the role of some genetic variants. Currently, there are no treatments for diffuse MCDs, while for focal malformations, which include FCD and HME, the treatment of choice is epilepsy surgery, which is however applicable only to a subset of patients. Recently, a series of clinical trials on patients and in vivo and in vitro studies in cellular and animal models have demonstrated that pharmacological rescue of PI3K/AKT/mTOR pathway dysregulation represents a novel promising treatment option for FCD/HME. In patients carrying mutations in mTOR pathway genes, the use of first generation mTOR inhibitors (known as rapalogs) might represent an example of personalized treatment based on the knowledge of the etiological cause of the disease. Although the use of rapalogs in various clinical trials have validated the concept that targeting PI3K/Akt/mTOR has a potential therapeutic effect, patients need treatment reduction or discontinuation due to severe adverse events. Metformin, a drug widely used to treat type 2 diabetes, has been recently shown to activate the AMP-activated protein kinase (AMPK) and inhibit the mTOR pathway, causing a reduction in protein synthesis and cellular proliferation. The aim of this PhD project has been to apply a multidisciplinary approach to identify causative mutations in two patients with MCDs, validate the pathogenic role of the identified mutations, and assess the effectiveness of metformin in rescuing the phenotype induced by one of the two mutations. The first mutation (Trp219Arg missense mutation in the LIS1 gene) was identified in a male patient (Patient 1) with delay of expressive language, intellectual and motor developments and pachygyria. The patient inherited the mutation from his affected mother. In literature, it has been demonstrated that missense substitutions in LIS1 have variable impact on the protein in terms of folding, protein–protein interactions, and stability and are linked to an equally variable phenotypic severity. For this reason, to explore if the mutation we observed impaired one of these processes, we performed a LIS1 gene expression study by qPCR, which detected no significant differences between proband, mother and controls and western blot analysis, which detected normal levels of the LIS1 protein in the proband and in his mother compared to controls. Therefore, the p.Trp219Arg mutation alters neither qualitative nor quantitative LIS1 expression and does not appear to cause defective folding and degradation of the mutant protein. These results suggest that the p.Trp219Arg mutation might instead cause mildly defective protein–protein interactions, resulting in a mild phenotype. They also suggest that different extents of impairment of this mechanism might contribute to the considerable phenotypic variability observed among individuals with LIS1 missense variants. The second mutation (p.Thr1977Ile missense mutation in the mTOR gene), was identified in a male patient (Patient 2) who presented profound intellectual disability, megalencephaly (+2.5 SD), cutaneous pigmentary mosaicism, bilateral frontal-parietal-insular polymicrogyria, and focal epilepsy. In our patient, the mutation was somatic and was present with different percentages of mosaicism in the different tissues we tested. Since we detected the mutation also in patient's fibroblasts, a cell line easily accessible, we tested the effectiveness of a novel potential mTOR pathway inhibitor in such cells. To confirm that the mutation affected the regulation of mTOR pathway in patient’s fibroblasts in response to nutrients deprivation, we evaluated the levels of P-RPS6 (direct downstream effector of mTOR protein) expression in cells incubated with a balanced salt solution used to induce starvation in fibroblasts. We observed an increased activation of the pathway caused by the gain-of-function effect of the mutation. In order to rescue of PI3K/AKT/mTOR pathway dysregulation induced by the p.Thr1977Ile mTOR mutation, we treated patient’s and control’s cells with metformin, demonstrating that this treatment is able to revert mTOR pathway hyperactivation. Overall, the results we obtained point to the growing need to associate NGS analysis with rapid and effective tests in order to validate the causative effect of the identified mutations. In addition, we demonstrated that for mTOR related MCDs, patients’ fibroblasts may represent a good model to test novel pharmacological treatments.