title: Functional complementation in yeast allows molecular characterization of missense argininosuccinate lyase mutations.
abstract: Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL(null) strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, "compound heterozygous" yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.
authors.affiliation: Clinical Genetics Unit, University of Padova, 35128 Padova, Italy.
authors.forename: Eva
authors.initials: E
authors.lastname: Trevisson
authors.forename: Alberto
authors.initials: A
authors.lastname: Burlina
authors.forename: Mara
authors.initials: M
authors.lastname: Doimo
authors.forename: Vanessa
authors.initials: V
authors.lastname: Pertegato
authors.forename: Alberto
authors.initials: A
authors.lastname: Casarin
authors.forename: Luca
authors.initials: L
authors.lastname: Cesaro
authors.forename: Placido
authors.initials: P
authors.lastname: Navas
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Basso
authors.forename: Geppo
authors.initials: G
authors.lastname: Sartori
authors.forename: Leonardo
authors.initials: L
authors.lastname: Salviati
mesh_terms: D000483:Alleles; D001123:Argininosuccinate Lyase / Q000235:genetics*; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D005838:Genotype; D006238:Haploidy; D006579:Heterozygote; D006801:Humans; D008957:Models, Genetic; D008968:Molecular Conformation; D009154:Mutation; D020125:Mutation, Missense; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014508:Urea / Q000378:metabolism
chemical_list: D014508:Urea; D001123:Argininosuccinate Lyase
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