Allelic phenotype values: a model for genotype-based phenotype prediction in phenylketonuria.

PURPOSE: The nature of phenylalanine hydroxylase (PAH) variants determines residual enzyme activity, which modifies the clinical phenotype in phenylketonuria (PKU). We exploited the statistical power of a large genotype database to determine the relationship between genotype and phenotype in PKU. METHODS: A total of 9336 PKU patients with 2589 different genotypes, carrying 588 variants, were investigated using an allelic phenotype value (APV) algorithm. RESULTS: We identified 251 0-variants encoding inactive PAH, and assigned APVs (0 = classic PKU; 5 = mild PKU; 10 = mild hyperphenylalaninaemia) to 88 variants in PAH-functional hemizygous patients. The genotypic phenotype values (GPVs) were set equal to the higher-APV allele, which was assumed to be dominant over the lower-APV allele and to determine the metabolic phenotype. GPVs for 8872 patients resulted in cut-off ranges of 0.0-2.7 for classic PKU, 2.8-6.6 for mild PKU and 6.7-10.0 for mild hyperphenylalaninaemia. Genotype-based phenotype prediction was 99.2% for classic PKU, 46.2% for mild PKU and 89.5% for mild hyperphenylalaninaemia. The relationships between known pretreatment blood phenylalanine levels and GPVs (n = 4217), as well as tetrahydrobiopterin responsiveness and GPVs (n = 3488), were significant (both P < 0.001). CONCLUSIONS: APV and GPV are powerful tools to investigate genotype-phenotype associations, and can be used for genetic counselling of PKU families.

Role of GTP-CHI links PAH and TH in melanin synthesis in silkworm, Bombyx mori.

In insects, pigment patterns are formed by melanin, ommochromes, and pteridines. Here, the effects of pteridine synthesis on melanin formation were studied using 4th instar larvae of a wild-type silkworm strain, dazao (Bombyx mori), with normal color and markings. Results from injected larvae and in vitro integument culture indicated that decreased activity of guanosine triphosphate cyclohydrolase I (GTP-CH I, a rate-limiting enzyme for pteridine synthesis), lowers BH4 (6R-l-erythro-5,6,7,8-tetrahydrobiopterin, a production correlated with GTP-CH I activity) levels and eliminates markings and coloration. The conversion of phenylalanine and tyrosine to melanin was prevented when GTP-CH I was inhibited. When BH4 was added, phenylalanine was converted to tyrosine, and the tyrosine concentration increased. Tyrosine was then converted to melanin to create normal markings and coloration. Decreasing GTP-CH I activity did not affect L-DOPA (3,4-l-dihydroxyphenylalanine). GTP-CH I affected melanin synthesis by generating the BH4 used in two key reaction steps: (1) conversion of phenylalanine to tyrosine by PAH (phenylalanine hydroxylase) and (2) conversion of tyrosine to L-DOPA by TH (tyrosine hydroxylase). Expression profiles of BmGTPCH Ia, BmGTPCH Ib, BmTH, and BmPAH in the integument were consistent with the current findings.

Foreign compounds and intermediary metabolism: sulfoxidation bridges the divide.

It is widely appreciated that as a xenobiotic travels through an organism and interacts with the biochemical machinery of a living system, it most probably will undergo a number of metabolic alterations usually leading to a cluster of differing chemical species. Indeed, the modern 'metabonomic' approach, where earlier studied drug metabolism profiles have been reassessed, has indicated that there are normally many more previously unrecognised minor metabolites, and when all such biotransformation products are considered, then their total number is legion. It is now being recognised also that the same metabolic alteration of a substrate, especially a xenobiotic substrate, may be catalysed by more than one enzyme and that the previously sacrosanct notion of an enzyme's 'substrate specificity' may well be inverted to read a substrate's 'enzyme preference'. The following brief article attempts to highlight another aspect where our general acceptance of the 'status quo' needs to be reconsidered. The conventionally acknowledged division between the collection of enzymes that undertake intermediary metabolism and the group of enzymes responsible for xenobiotic metabolism may be becoming blurred. It may well be a prudent time to reassess the current dichotomous view. Overcoming inertia, with a realignment of ideas or alteration of perception, may permit new concepts to emerge leading to a more profound understanding and hopefully eventual benefits for mankind.

Genotype-phenotype correlations analysis of mutations in the phenylalanine hydroxylase (PAH) gene.

The aims of our research were to define the genotype-phenotype correlations of mutations in the phenylalanine hydroxylase (PAH) gene that cause phenylketonuria (PKU) among the Israeli population. The mutation spectrum of the PAH gene in PKU patients in Israel is described, along with a discussion on genotype-phenotype correlations. By using polymerase chain reaction/denaturing high-performance liquid chromatography (PCR/dHPLC) and DNA sequencing, we screened all exons of the PAH gene in 180 unrelated patients with four different PKU phenotypes [classic PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP)]. In 63.2% of patient genotypes, the metabolic phenotype could be predicted, though evidence is also found for both phenotypic inconsistencies among subjects with more than one type of mutation in the PAH gene. Data analysis revealed that about 25% of patients could participate in the future in (6R)-L: -erythro-5, 6, 7, 8-tetrahydrobiopterin (BH4) treatment trials according to their mutation genotypes. This study enables us to construct a national database in Israel that will serve as a valuable tool for genetic counseling and a prognostic evaluation of future cases of PKU.

Phenylalanine 4-monooxygenase and the S-oxidation of S-carboxymethyl-L-cysteine by human cytosolic fractions.

The purpose of this investigation was to reaction phenotype the identity of the cytosolic enzyme responsible for the S-oxidation of S-carboxymethyl-L-cysteine (SCMC) in female human hepatic cytosolic fractions. The identity of this enzyme in the female Wistar rat hepatic cytosolic fraction was found to be phenylalanine 4-monooxygenase (PAH). In pooled female human hepatic cytosolic fractions the calculated K(m) and V(max) for substrate (SCMC) activated PAH was 16.22 +/- 11.31 mM and 0.87 +/- 0.41 nmoles x min(-1) mg(-1). The experimental data modelled to the Michaelis-Menten equation with noncompetitive substrate inhibition. When the cytosolic fractions were activated with lysophophatidylcholine the V(max) increased to 52.31 +/- 11.72 nmoles x min(-1) mg(-1) but the K(m) remained unchanged at 16.53 +/- 2.32 mM. A linear correlation was seen in the production of Tyr and SCMC R/S S-oxide in 20 individual female hepatic cytosolic fractions for both substrate and lysophosphatidylcholine activated PAH (r(s) > 0.96). Inhibitor studies found that the specific chemical and antibody inhibitors of PAH reduced the production of Tyr and SCMC R/S S-oxide in these in vitro PAH assays. An investigation of the mechanism of interaction of SCMC with PAH indicated that the drug was a competitive inhibitor of the aromatic C-oxidation of Phe with a calculated K(i) of 17.23 +/- 4.15 mM. The requirement of BH4 as cofactor and the lack of effect of the specific tyrosine hydroxylase, tryptophan hydroxylase and nitric oxide synthase inhibitors on the S-oxidation of SCMC all indicate that PAH was the enzyme responsible for this biotransformation reaction in human hepatic cytosolic fractions.

Elevated plasma phenylalanine in severe malaria and implications for pathophysiology of neurological complications.

Cerebral malaria is associated with decreased production of nitric oxide and decreased levels of its precursor, l-arginine. Abnormal amino acid metabolism may thus be an important factor in malaria pathogenesis. We sought to determine if other amino acid abnormalities are associated with disease severity in falciparum malaria. Subjects were enrolled in Dar es Salaam, Tanzania (children) (n = 126), and Papua, Indonesia (adults) (n = 156), in two separate studies. Plasma samples were collected from subjects with WHO-defined cerebral malaria (children), all forms of severe malaria (adults), and uncomplicated malaria (children and adults). Healthy children and adults without fever or illness served as controls. Plasma amino acids were measured using reverse-phase high-performance liquid chromatography with fluorescence detection. Several plasma amino acids were significantly lower in the clinical malaria groups than in healthy controls. Despite the differences, phenylalanine was the only amino acid with mean levels outside the normal range (40 to 84 microM) and was markedly elevated in children with cerebral malaria (median [95% confidence interval], 163 [134 to 193] microM; P < 0.0001) and adults with all forms of severe malaria (median [95% confidence interval], 129 [111 to 155] microM; P < 0.0001). In adults who survived severe malaria, phenylalanine levels returned to normal, with clinical improvement (P = 0.0002). Maintenance of plasma phenylalanine homeostasis is disrupted in severe malaria, leading to significant hyperphenylalaninemia. This is likely a result of an acquired abnormality in the function of the liver enzyme phenylalanine hydroxylase. Determination of the mechanism of this abnormality may contribute to the understanding of neurological complications in malaria.

A potential role for phenylalanine hydroxylase in mosquito immune responses.

In mosquitoes the melanotic encapsulation immune response is an important resistance mechanism against filarial worms and malaria parasites. The rate limiting substrate for melanin production is tyrosine that is hydroxylated by phenoloxidase (PO) to produce 3, 4-dihydroxyphenylalanine. The single pathway for endogenous production of tyrosine is by hydroxylation of phenylalanine by phenylalanine hydroxylase (PAH). In this study we describe a potential role for PAH in melanotic immune responses in the yellow fever mosquito, Aedes aegypti. A 1.6 kb A. aegypti PAH cDNA, encoding a 51 kDa protein, was isolated and subsequently expressed in an Escherichia coli expression system. In developing mosquitoes, PAH transcript is present in all stages and it is differentially expressed in adult tissues. Following an immune-challenge with Dirofilaria immitis microfilariae (mf) or bacteria, PAH transcript is up-regulated in hemocytes. Likewise, western analysis of hemocytes collected from immune-activated mosquitoes show an increase in gene product over control samples. Like PO, ultrastructure observations provide verification that PAH is located in oenocytoid and granulocyte hemocytes. Our results offer the first data that suggest PAH is used in mosquito melanin synthesis and defense responses.

How PAH gene mutations cause hyper-phenylalaninemia and why mechanism matters: insights from in vitro expression.

Mutations in the human PAH gene, which encodes phenylalanine hydroxylase are associated with varying degrees of hyperphenylalaninemia (HPA). The more severe of these manifest as a classic metabolic disease--phenylketonuria (PKU). In vitro expression analysis of PAH mutations has three major applications: 1) to confirm that a disease-associated mutation is genuinely pathogenic, 2) to assess the severity of a mutation's impact, and 3) to examine how a mutation exerts its deleterious effects on the PAH enzyme, that is, to elucidate the molecular mechanisms involved. Data on expression analysis of 81 PAH mutations in multiple in vitro systems is summarized in tabular form online at www.pahdb.mcgill.ca. A review of these findings points in particular to a prevalent general mechanism that appears to play a major role in the pathogenicity of many PAH mutations. Amino acid substitutions promote misfolding of the PAH protein monomer and/or oppose the correct assembly of monomers into the native tetrameric enzyme. The resulting structural aberrations trigger cellular defenses, provoking accelerated degradation of the abnormal protein. The intracellular steady-state levels of the mutant PAH enzyme are therefore reduced, leading to an overall decrease in phenylalanine hydroxylation within cells and thus to hyperphenylalaninemia. There is considerable scope for modulation of the enzymic and metabolic phenotypes by modification of the cellular handling--folding, assembly, and degradation--of the mutant PAH protein. This has major implications, both for our understanding of genotype-phenotype correlations and for the development of novel therapeutic approaches.

Phenylketonuria: genotype-phenotype correlations based on expression analysis of structural and functional mutations in PAH.

When analyzed in the context of the phenylalanine hydroxylase (PAH) three-dimensional structure, only a minority of the PKU mutations described world-wide affect catalytic residues. Consistent with these observations, recent data point to defective folding and subsequent aggregation/degradation as a predominant disease mechanism for several mutations. In this work, we use a combined approach of expression in eukaryotic cells at different temperatures and a prokaryotic system with co-expression of chaperonins to elucidate and confirm structural consequences for 18 PKU mutations. Three mutations are located in the amino terminal regulatory domain and 15 in the catalytic domain. Four mutations were found to abolish the specific activity in all conditions. Two are catalytic mutations (Y277D and E280K) and two are severe structural defects (IVS10-11G>A and L311P). All the remaining mutations (D59Y, I65T, E76G, P122Q, R158Q, G218V, R243Q, P244L, R252W, R261Q, A309V, R408Q, R408W, and Y414C) are folding defects causing reduced stability and accelerated degradation, although some of them probably affect residues involved in regulation. In these cases, we have demonstrated that the amount of mutant PAH protein and residual activity could be modulated by in vitro experimental conditions, and therefore the observed in vivo metabolic variation may be explained by interindividual variation in the quality control systems. The results derived provide an experimental framework to define the mutation severity relating genotype to phenotype. They also explain the observed inconsistencies for some mutations in patients with similar genotype and different phenotypes.

Tetrahydrobiopterin sensitivity in German patients with mild phenylalanine hydroxylase deficiency.

We report the results of tetrahydrobiopterin (BH4) loading tests in 10 German patients with mild phenylketonuria. A significant decline of phenylalanine values after application of BH4 was observed in all but one patients. Molecular genetic analyses revealed a range of different PAH gene mutations. Re-testing of one patient previously reported as non-responsive to BH4 loading showed a moderate response with a higher dose of BH4. Nevertheless, there appear to be kinetic differences in phenylalanine hydroxylation in patients with the same genotype. Non-responsiveness to 20 mg/kg BH4 was observed only in a single patient who was compound heterozygous for the novel mutation R176P (c.527G>C) and the common null-mutation P281L. In summary, our data are in line with recent reports indicating that BH4 sensitivity is a normal feature of most mild forms of PAH deficiency but may be influenced by other factors.

Structural characterization of the N-terminal autoregulatory sequence of phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is activated by its substrate phenylalanine, and through phosphorylation by cAMP-dependent protein kinase at Ser16 in the N-terminal autoregulatory sequence of the enzyme. The crystal structures of phosphorylated and unphosphorylated forms of the enzyme showed that, in the absence of phenylalanine, in both cases the N-terminal 18 residues including the phosphorylation site contained no interpretable electron density. We used nuclear magnetic resonance (NMR) spectroscopy to characterize this N-terminal region of the molecule in different stages of the regulatory pathway. A number of sharp resonances are observed in PAH with an intact N-terminal region, but no sharp resonances are present in a truncation mutant lacking the N-terminal 29 residues. The N-terminal sequence therefore represents a mobile flexible region of the molecule. The resonances become weaker after the addition of phenylalanine, indicating a loss of mobility. The peptides corresponding to residues 2-20 of PAH have different structural characteristics in the phosphorylated and unphosphorylated forms, with the former showing increased secondary structure. Our results support the model whereby upon phenylalanine binding, the mobile N-terminal 18 residues of PAH associate with the folded core of the molecule; phosphorylation may facilitate this interaction.

Assessing the relative importance of the biophysical properties of amino acid substitutions associated with human genetic disease.

The inclusion of a mutation in a pathology-based database such as the Human Gene Mutation Database (HGMD) is a two-stage process: first, the mutation must occur at the DNA level, then it must cause a clinically detectable disease state. The likelihood of the latter step, termed the relative clinical observation likelihood (RCOL), can be regarded as a function of the structural/functional consequences of a mutation at the protein level. Following this paradigm, we modeled in silico all amino acid replacements that could potentially have arisen from an inherited single base pair substitution in five human genes encoding arylsulphatase A (ARSA), antithrombin III (SERPINC1), protein C (PROC), phenylalanine hydroxylase (PAH), and transthyretin (TTR). These proteins were chosen on the basis of 1) the availability of a crystallographic structure, and 2) a sufficiently large number of amino acid replacements being logged in HGMD. A total of 9,795 possible mutant structures were modeled and 20 different biophysical parameters assessed. Together with the HGMD-derived spectra of clinically detected mutations, these data allowed maximum likelihood estimation of RCOL profiles for the 20 parameters studied. Nine parameters (including energy difference between wild-type and mutant structures, accessibility of the mutated residue, and distance from the binding/active site) exhibited statistically significant variability in their RCOL profiles, indicating that mutation-associated changes affected protein function. As yet, however, a biological meaning could only be attributed to the RCOL profiles of solvent accessibility and, for three proteins, local energy change, disturbed geometry, and distance from the active center. The limited ability of the biophysical properties of mutations to explain clinical consequences is probably due to our current lack of understanding as to which amino acid residues are critical for protein folding. However, since the proteins examined here were unrelated, and our findings consistent, it may nevertheless prove possible to extrapolate to other proteins whose dysfunction underlies inherited disease.

Estimation of amino acid pairs sensitive to variants in human phenylalanine hydroxylase protein by means of a random approach.

In this data-based theoretical analysis, we use a random approach to estimate amino acid pairs in human phenylalanine 4-hydroxylase (PAH) protein in order to determine which amino acid pairs are more sensitive to 187 variants in human PAH protein. The rationale of this study is based on our hypothesis and previous findings that the harmful variants are more likely to occur at randomly unpredictable amino acid pairs rather than at randomly predictable pairs. This is reasonable to argue as randomly predictable amino acid pairs are less likely to be deliberately evolved, whereas randomly unpredictable amino acid pairs are probably deliberately evolved in connection with protein function. 94.12% of 187 variants occurred at randomly unpredictable amino acid pairs, which accounted for 71.84% of 451 amino acid pairs in human PAH protein. The chance of a variant occurring is five times higher in randomly unpredictable amino acid pairs than in predictable pairs. Thus, randomly unpredictable amino acid pairs are more sensitive to variance in human PAH protein. The results also suggest that the human PAH protein has a natural tendency towards variants.

Mutagenesis of the regulatory domain of phenylalanine hydroxylase.

The regulatory domain of phenylalanine hydroxylase (PAH, EC ) consists of more than 100 amino acids at the N terminus, the removal of which significantly activates the enzyme. To study the regulatory properties controlled by the N terminus, a series of truncations and site-specific mutations were made in this region of rat PAH. These enzymes were expressed highly in Escherichia coli and purified through a pterin-conjugated Sepharose affinity column. The removal of the first 26 amino acids of the N terminus increased the activity by about 20-fold, but removal of the first 15 amino acids increased the activity by only 2-fold. Replacing serine-29 of rat PAH with cysteine from the same site of human PAH increased the activity by more than 4-fold. Mutation of serine to other amino acids with varying side chains: alanine, methionine, leucine, aspartic acid, asparagine, and arginine also resulted in significant activation, indicating a serine-specific inhibitory effect. But these site-specific mutants showed 30--40% lower activity when assayed with 6-methyl-5,6,7,8-tetrahydropterin. Stimulation of hydroxylase activity by preincubation of the enzyme with phenylalanine was inversely proportional to the activation state of all these mutants. Combined with recent crystal structures of PAH [Kobe, B. et al. (1999) Nat. Struct. Biol. 6, 442-448; and Erlandsen, H., Bjorgo, E., Flatmark, T. & Stevens, R. C. (2000) Biochemistry 39, 2208-2217], these data suggest that residues 16-26 have a controlling regulatory effect on the activity by interaction with the dihydroxypropyl side chain of (6R)-5,6,7,8-tetrahydrobiopterin. The serine/cysteine switch explains the difference in regulatory properties between human and rat PAH. The N terminus as a whole is important for maintaining rat PAH in an optimum catalytic conformation.

Monogenic traits are not simple: lessons from phenylketonuria.

The classification of genetic disease into chromosomal, monogenic and multifactorial categories is an oversimplification. Phenylketonuria (PKU) is a classic 'monogenic' autosomal recessive disease in which mutation at the human PAH locus was deemed sufficient to explain the impaired function of the enzyme phenylalanine hydroxylase (enzymic phenotype), the attendant hyperphenylalaninemia (metabolic phenotype) and the resultant mental retardation (cognitive phenotype). In the era of molecular genetics, expectations for a consistently close correlation between the mutant genotype and variant phenotype have been somewhat disappointed, and PKU is used here to illustrate how and why this might be the case. So-called monogenic traits do, indeed, conform to long-accepted ideas about the expression of 'major' loci and their importance in determining parameters of phenotype, but the associated features are as complex, in their own ways, as those in so-called complex traits.

A phenylalanine hydroxylase gene from the nematode C. elegans is expressed in the hypodermis.

We have identified an aromatic amino acid hydroxylase gene from the nematode C. elegans that likely encodes the worm phenylalanine hydroxylase (PheH). The predicted amino acid sequence is most similar to that of other PheH and TrpH proteins. Reporter gene fusions and staining with an antibody to mammalian PheH indicate the gene is expressed in hypodermal cells. A fusion protein expressed in bacteria can convert phenylalanine to tyrosine, and, to a lesser extent, tryptophan to 5-hydroxytryptophan. We hypothesize that the protein is necessary to produce additional tyrosine for protein cross-linking in the nematode cuticle.

In vitro expression analysis of mutations in phenylalanine hydroxylase: linking genotype to phenotype and structure to function.

Mutations in the human phenylalanine hydroxylase gene (PAH) altering the expressed cDNA nucleotide sequence (GenBank U49897) can impair activity of the corresponding enzyme product (hepatic phenylalanine hydroxylase, PAH) and cause hyperphenylalaninemia (HPA), a metabolic phenotype for which the major disease form is phenylketonuria (PKU; OMIM 261600). In vitro expression analysis of inherited human mutations in eukaryotic, prokaryotic, and cell-free systems is informative about the mechanisms of mutation effects on enzymatic activity and their predicted effect on the metabolic phenotype. Corresponding analysis of site-directed mutations in rat Pah cDNA has assigned critical functional roles to individual amino acid residues within the best understood species of phenylalanine hydroxylase. Data on in vitro expression of 35 inherited human mutations and 22 created rat mutations are reviewed here. The core data are accessible at the PAH Mutation Analysis Consortium Web site (http://www.mcgill.ca/pahdb).

Analysis of phenylalanine hydroxylase genotypes and hyperphenylalaninemia phenotypes using L-[1-13C]phenylalanine oxidation rates in vivo: a pilot study.

Hyperphenylalaninemia (HPA) resulting from deficient activity of phenylalanine hydroxylase (PAH) is caused by mutations in the human PAH gene (McKusick 261600). Herein, we report a noninvasive method to: 1) estimate whole-body phenylalanine oxidation in patients with HPA and 2) compare effects of mutant genotypes on phenotypes. We used oral L-[1-13C]phenylalanine as a substrate and measured 13CO2 formation in the first hour as an index of phenylalanine oxidation rates in: 1) patients with PKU (n = 6), variant phenylketonuria (PKU) (n = 7) and non-PKU HPA (n = 4); 2) obligate heterozygotes (n = 18); and 3) controls (n = 8). PAH mutations were identified by PCR, denaturing gradient gel electrophoresis, and DNA sequencing. Phenylalanine oxidation rates demonstrated a gene dosage effect; oxidation in heterozygotes was intermediate between probands and controls. The three classes of HPA had different mean oxidation rates (PKU < variant PKU < non-PKU HPA). The in vivo phenotype (HPA class or whole-body oxidation rate) did not always correspond to prediction from in vitro expression analysis of the mutation effect on enzyme activity. The findings indicate that the in vivo metrical trait (phenylalanine oxidation rate) is not a simple equivalent of phenylalanine hydroxylation activity (unit of protein phenotype) and, as expected, is an emergent property under the control of more than the PAH locus.

Reconstitution of enzymatic activity in hepatocytes of phenylalanine hydroxylase-deficient mice.

Phenylketonuria (PKU) is a metabolic disorder secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). The recent creation of a mouse strain for PAH deficiency has provided an excellent model system to explore the possibility of its phenotypic correction by hepatic gene therapy. A recombinant retrovirus containing the mouse PAH cDNA under the transcriptional control of the human CMV promoter was constructed and used to transduce hepatocytes isolated from PAH-deficient mice. Viral-transduced hepatocytes produced dramatically higher levels of mouse PAH mRNA as compared to control mock-infected hepatocytes. The PAH mRNA was translated efficiently into PAH protein that is capable of converting phenylalanine to tyrosine in vitro. These results demonstrate that the PAH-deficient mouse hepatocytes can be readily reconstituted by retroviral-mediated gene transduction, which is a crucial step towards somatic gene therapy for PKU.