Characterization of experimental phenylketonuria augmentation of hyperphenylalaninemia with α-methylphenylalanine and p-chlorophenylalanine

Phenylalanine in conjuction with p-chlorophenylalanine or α-methylphenylalanine was administered to suckling rats to induce hyperphenylalaninemia reminiscent of untreated phenylketonuria, and developmental parameters were monitored. The experimental model utilizing p-chlorophenylalanine was found to be unsatisfactory, in that the drug had general deleterous effects on growth, numerous side effects including increased mortality, and affected brain levels of biogenic monoamine neurotransmitters. The model utilizing α-methylphenylalalanine was relatively free from nonspecific effects and thus, changes observed in the animals were attributable to expereimental phenylketonuria. The latter animals had slightly decreased body and brain weights, and exhibited grossly elevated serum phenylalanine and urinary excretion of phenylketone metabolites. Hyperphenylalaninemia produced greatly disrupted brain amino acids at 10 days of age (prior to the formalization of the blood-brain barrier and specific transport systems) which was limited by 30 days of age to changes in glycine, γ-aminobutyric acid and the aliphatic and aromatic amino acids which compete for uptake in t he brain by a common carrier. These animals also exhibited a myelin deficit and changes in proteins from isolated nerve cell preparations. Mature animals which had daily treatment up to 60 days of age results obtained with animal models and the clinical findings in untreated phenylketonuric patients.     

Human Phenylalanine Hydroxylase Mutations and Hyperphenylalaninemia Phenotypes: A Metanalysis of Genotype-Phenotype Correlations

We analyzed correlations between mutant genotypes at the human phenylalanine hydroxylase locus (gene symbol PAH) and the corresponding hyperphenylalaninemia (HPA) phenotypes (notably, phenylketonuria [OMIM 261600]). We used reports, both published and in the PAH Mutation Analysis Consortium Database, on 365 patients harboring 73 different PAH mutations in 161 different genotypes. HPA phenotypes were classified as phenylketonuria (PKU), variant PKU, and non-PKU HPA. By analysis both of homoallelic mutant genotypes and of "functionally hemizygous" heteroallelic genotypes, we characterized the phenotypic effect of 48 of the 73 different, largely missense mutations. Among those with consistent in vivo expression, 24 caused PKU, 3 caused variant PKU, and 10 caused non-PKU HPA. However, 11 mutations were inconsistent in their effect: 9 appeared in two different phenotype classes, and 2 (I65T and Y414C) appeared in all three classes. Seven mutations were inconsistent in phenotypic effect when in vitro (unit-protein) expression was compared with the corresponding in vivo phenotype (an emergent property). We conclude that the majority of PAH mutations confer a consistent phenotype and that this is concordant with their effects, when known, predicted from in vitro expression analysis. However, significant inconsistencies, both between in vitro and in vivo phenotypes and between different individuals with similar PAH genotypes, reveal that the HPA-phenotype is more complex than that predicted by Mendelian inheritance of alleles at the PAH locus.     

Neopterin and Biopterin Levels in Patients with Atypical Forms of Phenylketonuria

The pattern of unconjugated pterins in liver tissue and in urine from patients with atypical forms of phenylketonuria with hyperphenylalaninemia (HPA) has been investigated with a high performance liquid chromatographic technique. Two patients with defects in the biosynthesis of biopterin have been shown to have higher than normal levels of neopterin and lower than normal levels of biopterin. In contrast, a patient with HPA due to a deficiency of dihydropteridine reductase has the reverse urinary pattern, i.e., high biopterin, low neopterin. These results indicate that the ratio of neopterin to biopterin in urine can be of value in discriminating between HPA due to a deficiency of phenylalanine hydroxylase (classic PKU), HPA due to dihydropteridine reductase deficiency, and HPA due to a block in the biosynthesis of biopterin.     

Effect of experimental hyperphenylalaninemia induced by dietary phenylalanine plus α-methylphenylalanine administration on amino acid concentration in neonatal chick brain,plasma, and liver

Supplementation of 5% phenylalanine plus 0.4% -methylphenylalanine to the standard diet or 1% phenylalanine plus 0.08% -methylphenylalanine to the drinking water produced phenylketonuria-like conditions in 5-day-old chicks. An increase of 10 to 15-fold in the phenylalanine content was observed in plasma or brain of animals after 9 days of both types of treatment. A smaller but significant increase was also observed in liver. However, practically no changes were found in the levels of tyrosine in the same conditions. Thus, the high values of plasma and brain phenylalanine/tyrosine ratio obtained by these treatments were mainly due to an increase in the phenylalanine levels, without increasing those of tyrosine. Chronic hyperphenylalaninemia induced a nonsignificant decrease in the most of amino acid contents in brain, especially after 9 days of treatment, although the levels of glycine and serine were significantly increased. A similar decrease was found in the plasma and liver concentration of various amino acids, although the variations observed in the liver were smaller than those found in plasma and brain.     

Chronic Hyperphenylalaninemia Produces Cerebral Hyperglycinemia in Immature Rats

The amino acid content of three tissues was measured in 10-day-old rats made hyperphenylalaninemic from age 3 to 10 days by daily injection of phenylalanine plus alpha-methylphenylalanine to inhibit phenylalanine hydroxylase (PAH). At 12 h after the last injection, the concentrations of alanine, valine, methionine, isoleucine, and leucine in the cerebral hemispheres were depressed by 25-50%, whereas that of glycine was elevated 2.3-fold. In the spinal cord, the levels of phosphoserine, methionine, and leucine were decreased by 40-50%, and those of serine and threonine increased by 50%. Tyrosine and phenylalanine concentrations were high in all tissues, 2-3 and 15-30 times normal, respectively; of the amino acids investigated, they were the only ones changed in the liver. Cerebral hyperglycinemia was also produced by chronic treatment with phenylalanine plus p-chlorophenylalanine to inhibit PAH, but not by acute (12 h) hyperphenylalaninemia. An increase in cerebral phosphoserine phosphatase activity was greater in rats treated with phenylalanine plus PAH inhibitor than with inhibitor alone. The content of brain glycine normally declines with age from birth to 15 days; this decrease was prevented by chronic hyperphenylalaninemia. Attempts to reduce the cerebral glycine content of the hyperphenylalaninemic rats were unsuccessful. However, one of the therapeutic protocols, methionine loading, may be useful because it increased the methionine and decreased the phenylalanine contents in the brain.     

Inhibition of brain and liver 3-hydroxy-3-methylglutaryl-CoA reductase and mevalonate-5-pyrophosphate decarboxylase in experimental hyperphenylalaninemia

Experimental hyperphenylalaninemia has been induced in 5-day-old chicks by dietary treatments with phenylalanine and -methylphenylalanine. An increase of nearly 8-fold in plasma Phe/Tyr ratio was found after 4 days of supplementation the standard diet with 5% phenylalanine plus 0.4% -methylphenylalanine. The increase in this ratio was about 13-fold after 9 days of the same treatment. Similar results were observed in brain and liver, although the increases were smaller than those found in plasma. Total body, brain and liver weight decreased after 9 days of treatment. Phenylalanine plus -methylphenylalanine administration to 5-day-old chicks produced a significant decrease in the 3-hydroxy-3-methylglutary-CoA reductase and mevalonate-5-pyrophosphate decarboxylase specific activities from both brain and liver. These results demonstrated for the first time that experimental hyperphenylalaninemia inhibited different enzyme activites directly implicated in the regulation of cholesterogenesis. Therefore, a reduced cholesterol synthesis in brain may evidenciate the theory of an impaired myelination leading to mental retardation in phenylketonuria patients.     

Biochemical Defect of the hph-1 Mouse Mutant Is a Deficiency in GTP-Cyclohydrolase Activity

A hyperphenylalaninemic mouse mutant, hph-1, has been identified in the progeny of mice treated with the mutagen ethylnitrosourea. Phenylalanine hydroxylase activity levels in mutant liver lysates are reduced relative to normal, but correction for the amount of enzyme protein present demonstrates that the specific activity of this enzyme is normal in mutant mice. Quinonoid-dihydropteridine reductase activity is also normal. GTP-cyclohydrolase activity levels are essentially absent early in life and greatly diminished later in life. This finding has significant implications for the study of catecholamine neurotransmitter synthesis because GTP-cyclohydrolase catalyzes an important step in the de novo synthesis of tetrahydrobiopterin, an enzyme cofactor required for the synthesis of 3,4-dihydroxyphenylalanine (DOPA) and serotonin.     

Spectrum of mutations in Lebanese patients with phenylalanine hydroxylase deficiency

Phenylketonuria is an autosomal recessive inborn error of metabolism resulting from phenylalanine hydroxylase deficiency. Genetic basis of phenylalanine hydroxylase deficiency has been reported in various European and Asian countries with few reports available in Arab populations of the Mediterranean region. This is the first pilot study describing phenotype and genotype of 23 Lebanese patients with phenylketonuria. 48% of the patients presented mainly with neurological signs at a mean age of 2 years 9 months, as newborn screening is not yet a nationwide policy. 56.5% of the patients had classical phenylketonuria. Thirteen different mutations were identified: splice site 52%, frameshift 31%, and missense 17% with no nonsense mutations. IVS10-11G>A was found mainly in Christians at high relative frequency whereas Muslims carried the G352fs and R261Q mutations. A rare splice mutation IVS7+1G>T, not described before, was identified in the homozygous state in one family with moderate phenylketonuria phenotype. Genotype–phenotype correlation using Guldberg arbitrary value method showed high consistency between predicted and observed phenotypes. Calculated homozygosity rate was 0.07 indicating the genetic heterogeneity in our patients. Our findings underline the admixture of different ethnicities and religions in Lebanon that might help tracing back the PAH gene flux history across the Mediterranean region.     

Retrovirus-Mediated Double Transduction of the GTPCH and PTPS Genes Allows 6-Pyruvoyltetrahydropterin Synthase-Deficient Human Fibroblasts to Synthesize and Release Tetrahydrobiopterin

Abstract: The tetrahydrobiopterin (BH₄) cofactor is essential for the aromatic amino acid hydroxylases that are involved in phenylalanine degradation and catecholamine and serotonin biosynthesis. Furthermore, BH₄ is an essential and limiting cofactor for all types of nitric oxide synthases. BH₄ deficiency results in hyperphenylalaninemia and monoamine neurotransmitter depletion associated with progressive mental retardation and is most commonly due to autosomal recessive mutations in 6-pyruvoyltetrahydropterin synthase (PTPS), the second enzyme for cofactor biosynthesis. Due to the relatively poor blood-brain barrier penetration of the cofactor, conventional therapy requires, besides oral doses of synthetic BH₄, administration of neurotransmitter precursors and an aromatic amino acid decarboxylase inhibitor. The outcome of this therapy is not always beneficial. In this study we transduced into primary patient fibroblasts the human cDNAs for the BH₄ biosynthetic enzymes GTP cyclohydrolase I and PTPS, expressed from different retroviral vectors. This allowed BH₄ biosynthesis in originally PTPS-deficient cells. Moreover, the double-transduced fibroblasts released between 200 and 800 pmol of BH₄/10⁶ cells/day. Such engineered fibroblasts may be grafted into the central nervous system and used as depository cells for constitutive delivery of BH₄.