A Yeast-Based Repurposing Approach for the Treatment of Mitochondrial DNA Depletion Syndromes Led to the Identification of Molecules Able to Modulate the dNTP Pool.

Mitochondrial DNA depletion syndromes (MDS) are clinically heterogenous and often severe diseases, characterized by a reduction of the number of copies of mitochondrial DNA (mtDNA) in affected tissues. In the context of MDS, yeast has proved to be both an excellent model for the study of the mechanisms underlying mitochondrial pathologies and for the discovery of new therapies via high-throughput assays. Among the several genes involved in MDS, it has been shown that recessive mutations in MPV17 cause a hepatocerebral form of MDS and Navajo neurohepatopathy. MPV17 encodes a non selective channel in the inner mitochondrial membrane, but its physiological role and the nature of its cargo remains elusive. In this study we identify ten drugs active against MPV17 disorder, modelled in yeast using the homologous gene SYM1. All ten of the identified molecules cause a concomitant increase of both the mitochondrial deoxyribonucleoside triphosphate (mtdNTP) pool and mtDNA stability, which suggests that the reduced availability of DNA synthesis precursors is the cause for the mtDNA deletion and depletion associated with Sym1 deficiency. We finally evaluated the effect of these molecules on mtDNA stability in two other MDS yeast models, extending the potential use of these drugs to a wider range of MDS patients.

Pathogenic variants in glutamyl-tRNAGln amidotransferase subunits cause a lethal mitochondrial cardiomyopathy disorder.

Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex.

Pathological alleles of MPV17 modeled in the yeast Saccharomyces cerevisiae orthologous gene SYM1 reveal their inability to take part in a high molecular weight complex.

Mitochondrial DNA depletion syndromes (MDDS) are a genetically and clinically heterogeneous group of human diseases caused by mutations in nuclear genes and characterized by a severe reduction in mitochondrial DNA (mtDNA) copy number leading to impaired energy production in affected tissues and organs. Mutations in the MPV17 gene, whose role is still elusive, were described as cause of the hepatocerebral form of MDDS and Navajo neuro-hepathopathy. The high degree of conservation observed between MPV17 and its yeast homolog SYM1 made the latter a good model for the study of the pathology. Here, we used Saccharomyces cerevisiae to elucidate the molecular consequences of seven MPV17 missense mutations identified in patients and localized in different protein domains. The phenotypic analysis of the appropriate sym1 mutant strains created demonstrated deleterious effect for all mutations regarding OXPHOS metabolism and mtDNA stability. We deepened the pathogenic effect of the mutations by investigating whether they prevented the correct protein localization into the mitochondria or affected the stability of the proteins. All the Sym1 mutant proteins correctly localized into the mitochondria and only one mutation predominantly affects protein stability. All the other mutations compromised the formation of the high molecular weight complex of unknown composition, previously identified both in yeast, cell cultures and mouse tissues, as demonstrated by the consistent fraction of the Sym1 mutant proteins found free or in not fully assembled complex, strengthening its role as protein forming part of a high molecular weight complex.

Clinical Features, Molecular Heterogeneity, and Prognostic Implications in YARS2-Related Mitochondrial Myopathy.

Importance: YARS2 mutations have been associated with a clinical triad of myopathy, lactic acidosis, and sideroblastic anemia in predominantly Middle Eastern populations. However, the identification of new patients expands the clinical and molecular spectrum of mitochondrial disorders. Objectives: To review the clinical, molecular, and genetic features of YARS2-related mitochondrial disease and to demonstrate a new Scottish founder variant. Design, Setting, and Participants: An observational case series study was conducted at a national diagnostic center for mitochondrial disease in Newcastle upon Tyne, England, and review of cases published in the literature. Six adults in a well-defined mitochondrial disease cohort and 11 additional cases described in the literature were identified with YARS2 variants between January 1, 2000, and January 31, 2015. Main Outcome and Measures: The spectrum of clinical features and disease progression in unreported and reported patients with pathogenic YARS2 variants. Results: Seventeen patients (median [interquartile range] age at onset, 1.5 [9.8] years) with YARS2-related mitochondrial myopathy were identified. Fifteen individuals (88%) exhibited an elevated blood lactate level accompanied by generalized myopathy; only 12 patients (71%) manifested with sideroblastic anemia. Hypertrophic cardiomyopathy (9 [53%]) and respiratory insufficiency (8 [47%]) were also prominent clinical features. Central nervous system involvement was rare. Muscle studies showed global cytochrome-c oxidase deficiency in all patients tested and severe, combined respiratory chain complex activity deficiencies. Microsatellite genotyping demonstrated a common founder effect shared between 3 Scottish patients with a p.Leu392Ser variant. Immunoblotting from fibroblasts and myoblasts of an affected Scottish patient showed normal YARS2 protein levels and mild respiratory chain complex defects. Yeast modeling of novel missense YARS2 variants closely correlated with the severity of clinical phenotypes. Conclusions and Relevance: The p.Leu392Ser variant is likely a newly identified founder YARS2 mutation. Testing for pathogenic YARS2 variants should be considered in patients presenting with mitochondrial myopathy, characterized by exercise intolerance and muscle weakness even in the absence of sideroblastic anemia irrespective of ethnicity. Regular surveillance and early treatment for cardiomyopathy and respiratory muscle weakness is advocated because early treatment may mitigate the significant morbidity and mortality associated with this genetic disorder.