Il lievito Saccharomyces cerevisiae: un modello per lo studio di mutazioni associate a neuropatie ottiche ereditarie (HON))

The hereditary optic neuropathies (HON) comprise a group of disorders that are characterized by selective loss of retinal ganglion cells (RGCs), leading to optic nerve atrophy and different degrees of visual impairment and blindness. Clinical variability often makes recognition and classification difficult. Traditionally, classification has relied on the recognition of similar characteristics and similar patterns of transmission, but genetic analysis now permits the specific diagnosis. Thanks to next generation sequencing thirteen mutations were identified. The yeast Saccharomyces cerevisiae is extensively used as a genetic system to study mitochondrial diseases, giving the possibility to prove with high confidence the link between novel mutations and mitochondrial pathologies, since yeast can survive without mitochondrial DNA and by fermenting. Once the mutant strains were constructed, the pathogenicity of these variants were demonstrated exploiting the analysis of the OXPHOS metabolism. In particular, it was shown that all the variants identified in patients led to a reduction in both oxidative growth and respiration, allowing to connect the pathological patients’ phenotype with the presence of the substitutions. Mild and severe phenotypes were observed, and to better understand the mechanisms underlying the disease, a series of molecular analyzes have been performed. Investigation on the enzymatic activity, in the SDHA and ACO2 mutant strains, highlighted a positive correlation with the results obtained in the OXPHOS metabolism analysis with that of the enzymatic capacity, suggesting that the expression of the pathological phenotype could be due to an enzymatic deficiency. For the other mutants, containing MTFMT and MECR, a series of analysis were performed. In MECR strains, a deficiency in lipoic acid was observed, since the mitochondrial fatty acids biosynthesis pathway is blocked. Lipoic acid is an essential cofactor for some of the enzymes important for the Krebs cycle, therefore a reduction in lipoic acid content was compatible with the oxidative defects observed. On the other hand, MTFMT, responsible for the formylation of the met-tRNAmet led to defects in mitochondrial protein synthesis which could explaining the pathological phenotype in patients. Furthermore, since ACO2 and SDHA mutations were in heterozygous condition, inheritance studies have been performed and, the diploid condition recreated in yeast, allowed to detect the pathological defect. In almost all the mutants, the dominance was confirmed, explaining that with haploinsufficiency or negative dominance. The results showed that yeast is an excellent tool for validation but also for extrapolating information on the inheritance of mutations.