Functional and structural analyses of novel Smith-Kingsmore Syndrome-Associated MTOR variants reveal potential new mechanisms and predictors of pathogenicity

Smith-Kingsmore syndrome (SKS) is a rare neurodevelopmental disorder characterized by macrocephaly/megalencephaly, developmental delay, intellectual disability, hypotonia, and seizures. It is caused by dominant missense mutations in MTOR. The pathogenicity of novel variants in MTOR in patients with neurodevelopmental disorders can be difficult to determine and the mechanism by which variants cause disease remains poorly understood. We report 7 patients with SKS with 4 novel MTOR variants and describe their phenotypes. We perform in vitro functional analyses to confirm MTOR activation and interrogate disease mechanisms. We complete structural analyses to understand the 3D properties of pathogenic variants. We examine the accuracy of relative accessible surface area, a quantitative measure of amino acid side-chain accessibility, as a predictor of MTOR variant pathogenicity. We describe novel clinical features of patients with SKS. We confirm MTOR Complex 1 activation and identify MTOR Complex 2 activation as a new potential mechanism of disease in SKS. We find that pathogenic MTOR variants disproportionately cluster in hotspots in the core of the protein, where they disrupt alpha helix packing due to the insertion of bulky amino acid side chains. We find that relative accessible surface area is significantly lower for SKS-associated variants compared to benign variants. We expand the phenotype of SKS and demonstrate that additional pathways of activation may contribute to disease. Incorporating 3D properties of MTOR variants may help in pathogenicity classification. We hope these findings may contribute to improving the precision of care and therapeutic development for individuals with SKS.
Author summary Smith-Kingsmore Syndrome is a rare disease caused by damage in a gene named MTOR that is associated with excessive growth of the head and brain, delays in development and deficits in intellectual functioning. We report 7 patients who have changes in MTOR that have never been reported before. We describe new medical findings in these patients that may be common in Smith-Kingsmore Syndrome more broadly. We then identify how these new gene changes impact the function of the MTOR protein and thus cell function downstream. Lastly, we show that changes in the gene that lie deep inside the 3D structure of the MTOR protein are more likely to cause disease than those changes that lie on the surface of the protein. We may be able to use the 3D properties of MTOR gene changes to predict if future changes we see are likely to cause disease or not.