title: Phenylketonuria: genotype-phenotype correlations based on expression analysis of structural and functional mutations in PAH.
abstract: When analyzed in the context of the phenylalanine hydroxylase (PAH) three-dimensional structure, only a minority of the PKU mutations described world-wide affect catalytic residues. Consistent with these observations, recent data point to defective folding and subsequent aggregation/degradation as a predominant disease mechanism for several mutations. In this work, we use a combined approach of expression in eukaryotic cells at different temperatures and a prokaryotic system with co-expression of chaperonins to elucidate and confirm structural consequences for 18 PKU mutations. Three mutations are located in the amino terminal regulatory domain and 15 in the catalytic domain. Four mutations were found to abolish the specific activity in all conditions. Two are catalytic mutations (Y277D and E280K) and two are severe structural defects (IVS10-11G>A and L311P). All the remaining mutations (D59Y, I65T, E76G, P122Q, R158Q, G218V, R243Q, P244L, R252W, R261Q, A309V, R408Q, R408W, and Y414C) are folding defects causing reduced stability and accelerated degradation, although some of them probably affect residues involved in regulation. In these cases, we have demonstrated that the amount of mutant PAH protein and residual activity could be modulated by in vitro experimental conditions, and therefore the observed in vivo metabolic variation may be explained by interindividual variation in the quality control systems. The results derived provide an experimental framework to define the mutation severity relating genotype to phenotype. They also explain the observed inconsistencies for some mutations in patients with similar genotype and different phenotypes.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
authors.forename: Angel L
authors.initials: AL
authors.lastname: Pey
authors.forename: Lourdes R
authors.initials: LR
authors.lastname: Desviat
authors.forename: Alejandra
authors.initials: A
authors.lastname: Gámez
authors.forename: Magdalena
authors.initials: M
authors.lastname: Ugarte
authors.forename: Belén
authors.initials: B
authors.lastname: Pérez
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D019295:Computational Biology / Q000379:methods; D003198:Computer Simulation; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics* / Q000502:physiology; D005838:Genotype; D006801:Humans; D051379:Mice; D009154:Mutation; D010641:Phenotype; D010651:Phenylalanine Hydroxylase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D010661:Phenylketonurias / Q000201:enzymology* / Q000235:genetics*; D017510:Protein Folding; D020836:Protein Structure, Quaternary / Q000235:genetics / Q000502:physiology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D013329:Structure-Activity Relationship
chemical_list: D011993:Recombinant Fusion Proteins; D010651:Phenylalanine Hydroxylase