OBJECTIVETo understand the pathogenic effect and the correlation between the genotype and phenotype of the 4 novel missense mutations (G247S, E280G, P362T and A434D) of phenylalanine hydroxylase gene (PAH).

METHODS(1) The enzyme activity of the 4 mutants was assessed by using transient protein expression in mammalian cells. (2) The PAH amino acid sequences among different animal species were alignmented. (3) The effects of the 4 missense mutations on the protein structure were analyzed. (4) The clinical phenotype of the patients with PKU were analyzed, according to their blood Phe levels prior to treatment and the Phe tolerance.

RESULTS(1) The residual enzyme activity expressed in vitro of G247S, E280G, P362T and A434D were 3.1%, 0.4%, 8.2% and 21.7% of the wild-type PAH respectively; (2)Gly247, Glu280 and Pro362 were among the highly conserved amino acids, while Ala434 was only moderately conserved; (3) As revealed by 3D structural analysis, G247S and E280G, being located at the active center of the enzyme, interfered with the binding of PAH to BH4 and ferrousion respectively, while P362T and A434D affected the formation and stability of the dimer and the tetramer of PAH; (4) As shown by clinical phenotypic analysis, classical PKU were observed in patients carrying G247S and E280G, moderate PKU were observed in patients carrying A434D, whereas both classical and moderate PKU were observed in patients carrying P362T.

CONCLUSION(1) The E280G, G247S, P362T and A434D are all disease-causing mutations, with those located at the center of the enzyme displaying the most marked pathogenic effect; (2)The results of the structural analysis of the 3D molecule are consistent with the activity assessment of the enzyme expressed in vitro; (3) The consistency is observed between the genotype, the enzymatic activity expressed in vitro and the clinical phenotype.