SVILUPPO DI MODELLI DI LIEVITO PER LA VALIDAZIONE DI NUOVE MUTAZIONI PATOGENICHE ASSOCIATE AD ANEURISMA DELL'AORTA TORACICA E DEFICIT PRIMARIO DI COENZIMA Q10

The yeast Saccharomyces cerevisiae is a cheap, time-saving and versatile tool to study human genetic diseases, since a great number of genes and biological processes are evolutionarily conserved. Moreover, residues at which mutations occur in human are often conserved in yeast. Not least, yeast can grow either by mitochondrial-dependent respiration or by ethanol fermentation, thus permitting to study mitochondrial diseases. We employed this model organism to test the functional effect of novel α-smooth muscle actin (ACTA2) heterozygous mutations, found in patients with familial thoracic aortic aneurysm and/or dissection (TAAD) and novel COQ4 autosomal recessive mutations associated with primary CoQ10 deficiency. ACTA2 is a major component of thin filaments of the contraction units of smooth muscle cells (SMCs). Polymerized fibers of ACTA2 filaments interact with myosin, regulating blood pressure and flow, and maintaining the structural integrity of blood vessels. Autosomal dominant missense mutations in the ACTA2 gene are the most common cause of familial non-syndromic TAAD, responsible for 14% of the cases. However, ACTA2 mutations are inherited with reduced penetrance (about 0,5) and variable expressivity with respect to the age of onset and the degree of aortic dilation. In addition, TAAD-associated ACTA2 genetic defects are thought to act by a dominant-negative mechanism and not through haploinsufficiency. Hence the importance of developing a system to functionally validate variants identified during patients screening. Here we report the development of a yeast model that allowed us to validate the pathogenicity of five novel human ACTA2 missense variants, found in TAAD patients. Furthermore, our data proved in vivo that TAAD-associated mutant actin isoforms exert a dominant negative effect on wild type actin, impairing its functionality. Next, we analysed COQ4 mutations found in patients suffering from primary CoQ10 deficiency, a rare mitochondrial disease caused by mutations in COQ genes, involved in Coenzyme Q biosynthesis. Clinical phenotypes of patients range from a fatal multisystem disease to isolated steroid resistant nephrotic syndrome or isolated central nervous system disease. In yeast and in mammalian cells, COQ proteins have been well characterized, but the role of some of them is not completely clear yet. For example, the precise function of COQ4 is unknown, even if it seems to have a key structural role for the formation and stability of the Coq polypeptide complex assembly, necessary for CoQ biosynthesis. Mutations in the COQ4 gene in most cases cause a lethal neonatal mitochondrial encephalomyopathy, but the clinical phenotype of patients and the age of onset vary widely, making diagnosis more difficult. By means of a recombinant yeast model we validated the pathogenicity of all tested COQ4 mutations; moreover, our findings support the putative central role of the COQ4 polypeptide in CoQ10 production. Yeast based assays described here provide a simple tool for validating new putative pathogenic ACTA2 or COQ4 variants, in order to perform a prompt diagnosis and institute a treatment as early as possible.