Yeast-based functional investigation of WARS2 variants and amino acid supplementation as a therapeutic strategy

Background/objectives WARS2 encodes the mitochondrial tryptophanyl-tRNA synthetase required for mitochondrial protein synthesis. Biallelic variants in WARS2 have been associated with a wide phenotypic spectrum, ranging from mitochondrial neurodevelopmental disorder with abnormal movements and lactic acidosis to early-onset dystonia-parkinsonism. Recently, several WARS2 pedigrees and variants have been identified, and previously reported cases reviewed within an expanding international study. Our cohort currently includes 104 affected individuals, including 62 new cases. These carry compound heterozygous or homozygous WARS2 variants, for a total of 64 mutant alleles, of which 41 are novel. Two mutational hotspots were identified. To define functional impairment and delineate the contribution of each WARS2 variant, while exploring genotype–phenotype correlations, we modelled selected missense variants from different protein domains in Saccharomyces cerevisiae. Methods Seven mutations were introduced into the yeast ortholog MSW1. Using a plasmid shuffling strategy in an msw1Δ background, we assessed oxidative growth, oxygen consumption rate (OCR), mitochondrial protein synthesis (MPS), and the effect of tryptophan supplementation. Results Functional analyses classified the variants into two amorphic and five hypomorphic alleles, the latter retaining partial mitochondrial activity. Among hypomorphic variants, a spectrum of functional severity was observed, ranging from severe impairment of oxidative growth, OCR, and MPS to milder defects. Importantly, some hypomorphic variants showed functional improvement upon tryptophan supplementation, indicating partial metabolic rescue. Conclusion Yeast modelling enabled rapid functional characterization of WARS2 variants and revealed allele-specific differences in functional severity. The responsiveness of selected hypomorphic alleles suggests amino acid supplementation may represent a variant-informed therapeutic strategy in selected patients.