RESUMEN
Hyperphenylalaninemia (Online Mendelian Inheritance in Man database: 261600) is an autosomal recessive disorder mainly due to mutations in the gene for phenylalanine hydroxylase; the most severe form of hyperphenylalaninemia is classic phenylketonuria. We sequenced the entire gene for phenylalanine hydroxylase in 51 unrelated hyperphenylalaninemia patients from Southern Italy. The entire locus was genotyped in 46 out of 51 hyperphenylalaninemia patients, and 32 different disease-causing mutations were identified. The pathologic nature of two novel gene variants, namely, c.707-2delA and p.Q301P, was demonstrated by in vitro studies. c.707-2delA is a splicing mutation that involves the accepting site of exon 7; it causes the complete skipping of exon 7 and results in the truncated p.T236MfsX60 protein. The second gene variant, p.Q301P, has very low residual enzymatic activity (approximately 4.4%), which may be ascribed, in part, to a low expression level (8-10%). Both the decreased enzyme activity and the low expression level are supported by analysis of the 3D structure of the molecule. The putative structural alterations induced by p.Q301P are compatible with protein instability and perturbance of monomer interactions within dimers and tetramers, although they do not affect the catalytic site. In vivo studies showed tetrahydrobiopterin responsiveness in the p.Q301P carrier but not in the c.707-2delA carrier. We next investigated genotype-phenotype correlations and found that genotype was a good predictor of phenotype in 76% of patients. However, genotype-phenotype discordance occurred in approximately 25% of our patients, mainly those bearing mutations p.L48S, p.R158Q, p.R261Q and p.P281L.
Asunto(s)
Genotipo , Mutación , Fenotipo , Fenilalanina Hidroxilasa/deficiencia , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Niño , Preescolar , Dihidropteridina Reductasa/genética , Femenino , Humanos , Italia/epidemiología , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Fenilalanina Hidroxilasa/química , Fenilcetonurias/metabolismo , Conformación ProteicaRESUMEN
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.