Functional analysis of human GCH1 variants using Saccharomyces cerevisiae as a model system

The GCH1 gene encodes GTP cyclohydrolase 1 (GTP-CH1), the first and rate-limiting enzyme in the biosynthesis of tetrahydrobiopterin, an essential cofactor in neurotransmitter synthesis. Pathogenetic variants in GCH1 are linked to malignant hyperphenylalaninemia, dopa-responsive dystonia (DRD), Parkinson’s disease (PD) and depression. Recently, novel missense GCH1 variants were identified in patients with DRD or PD. This study aims to investigate their potential pathogenicity using Saccharomyces cerevisiae as a model organism. In yeast, the FOL2 gene encodes GTP-CH1, essential for folate synthesis. The folinic acid auxotrophy of the fol2Δ strain can be rescued by human GCH1 cDNA, enabling a heterologous complementation approach. To assess pathogenicity, the novel variants were introduced into the human GCH1 cDNA and expressed in haploid strain lacking endogenous GTP-CH1 activity. We investigated their ability to rescue growth in folinate-free media and to utilize oxidative carbon sources. Mutants were classified as null or hypomorphic, i.e. exhibiting a heat-sensitive growth defect. Since these variants are heterozygous in patients, diploid strains were generated to mimic patients’ genetic context. Methotrexate was added to exacerbate growth defects, as hemiallelic strains are more sensitive to this compound than homoallelic strains. Overall, our results support a pathogenic role for most GCH1 variants analysed and provide insight into their molecular mechanisms.