title: Five human phenylalanine hydroxylase proteins identified in mild hyperphenylalaninemia patients are disease-causing variants.
abstract: Hyperphenylalaninemia is a group of autosomal recessive disorders caused by a wide range of phenylalanine hydroxylase (PAH) gene variants. To study the effects of mutations on PAH activity, we have reproduced five mutations (p.N223Y, p.R297L, p.F382L, p.K398N and p.Q419R) that we recently identified in a population of Southern Italy. Transient expression of mutant full-length cDNAs in human HEK293 cells yielded PAH variants whose l-phenylalanine hydroxylase activity was between 40% and 70% that of the wild-type enzyme. Moreover, Western blot analysis revealed a 50-kD monomer in all mutants thereby indicating normal synthesis of the mutant proteins. Because of the clinical mild nature of the phenotypes we performed an in vivo BH4 loading test. This was positive in all tested patients, which indicates that they are likely to respond to the coenzyme in vivo. We also analysed the environment of each mutation site in the available crystal structures of PAH by using molecular graphics tools. The structural alteration produced by each mutation was elucidated and correlated to the mutated properties of the mutant enzymes. All the data obtained demonstrate the disease-causing nature of the five novel variants.
authors.affiliation: CEINGE - Biotecnologie Avanzate Scarl, Via Comunale Margherita, 482, 80145, Naples, Italy.
authors.forename: Aurora
authors.initials: A
authors.lastname: Daniele
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Cardillo
authors.forename: Cinzia
authors.initials: C
authors.lastname: Pennino
authors.forename: Maria T
authors.initials: MT
authors.lastname: Carbone
authors.forename: Domenico
authors.initials: D
authors.lastname: Scognamiglio
authors.forename: Luciana
authors.initials: L
authors.lastname: Esposito
authors.forename: Antonio
authors.initials: A
authors.lastname: Correra
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Castaldo
authors.forename: Adriana
authors.initials: A
authors.lastname: Zagari
authors.forename: Francesco
authors.initials: F
authors.lastname: Salvatore
mesh_terms: D015153:Blotting, Western; D002460:Cell Line; D004252:DNA Mutational Analysis; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D006801:Humans; D007558:Italy; D008958:Models, Molecular; D009154:Mutation; D010651:Phenylalanine Hydroxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010661:Phenylketonurias / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D017433:Protein Structure, Secondary; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D010651:Phenylalanine Hydroxylase