Saccharomyces cerevisiae as a tool to assess the pathogenicity of human GCH1 variants

Background and objectives: The GCH1 gene encodes GTP cyclohydrolase 1, the first and rate-limiting enzyme in tetrahydrobiopterin biosynthesis, an essential cofactor for neurotransmitter synthesis. Pathogenic variants in GCH1 are linked to malignant hyperphenylalaninemia, dopa-responsive dystonia (DRD), Parkinson’s disease (PD) and depression. Recently, novel GCH1 variants were identified in patients with DRD or PD. To investigate their pathogenic potential, we modelled these variants in Saccharomyces cerevisiae. In yeast, the orthologous gene FOL2 is essential for folate biosynthesis; its impairment leads to folinic acid auxotrophy and affects one-carbon metabolism, consequently impairing oxidative phosphorylation. Methods: The auxotrophy of the fol2Δ strain can be rescued by expression of human GCH1; therefore, novel variants were introduced into GCH1 and expressed in a fol2Δ background. Growth on folinate-free medium and on respiratory carbon sources was assessed. Diploid strains modelling the patients’ heterozygous state were generated, and oxygen consumption rate (OCR) was measured. Results: Variants were classified as null or hypomorphic based on folinate auxotrophy and oxidative growth defect. In diploid strains, combined mutations resulted in reduced oxidative growth and OCR value compared to the control strain, indicating partial mitochondrial impairment. Conclusions: Our yeast model provides a robust in vivo system to assess the functional impact of GCH1 variants. By integrating folinate auxotrophy, oxidative growth, and respiratory measurements in haploid and diploid contexts, we defined the individual contribution of each variant and evaluated the effects of allelic combinations. These findings highlight yeast as a powerful tool to dissect variant pathogenicity and provide mechanistic insight into disease.