Five human phenylalanine hydroxylase proteins identified in mild hyperphenylalaninemia patients are disease-causing variants.
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.
Daniele
Aurora
A
CEINGE - Biotecnologie Avanzate Scarl, Via Comunale Margherita, 482, 80145, Naples, Italy.
Cardillo
Giuseppe
G
Pennino
Cinzia
C
Carbone
Maria T
MT
Scognamiglio
Domenico
D
Esposito
Luciana
L
Correra
Antonio
A
Castaldo
Giuseppe
G
Zagari
Adriana
A
Salvatore
Francesco
F
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
D010651:Phenylalanine Hydroxylase