PAHdb: a locus-specific knowledgebase

PAHdb is an online relational locus-specific "mutation database" (http://www.mcgill.ca/pahdb) for the human phenylalanine hydroxylase gene (symbol PAH) and its associated phenotypes (protein, metabolic, clinical). When combined with associated information (population distribution of allele, haplotype association, etc.) PAHdb functions as a knowledgebase. From the outset, and in the absence of raw data (e.g., sequence gels), PAHdb has instead been an annotated repository of information about mutations maintained by a team of curators. It is also disease-oriented, being focused on a variant phenotype (hyperphenylalaninemia (HPA) and its most important form of disease, phenylketonuria (PKU)) resulting from primary dysfunction of the PAH enzyme (EC 1.14.16.1); it is "patient friendly" in that it contains information for those personally involved with HPA/PKU (MIM# 261600). PAHdb also serves its community through direct interaction.     

PAHdb 2003: what a locus-specific knowledgebase can do

PAHdb, a legacy of and resource in genetics, is a relational locus-specific database (http://www.pahdb.mcgill.ca). It records and annotates both pathogenic alleles (n = 439, putative disease-causing) and benign alleles (n = 41, putative untranslated polymorphisms) at the human phenylalanine hydroxylase locus (symbol PAH). Human alleles named by nucleotide number (systematic names) and their trivial names receive unique identifier numbers. The annotated gDNA sequence for PAH is typical for mammalian genes. An annotated gDNA sequence is numbered so that cDNA and gDNA sites are interconvertable. A site map for PAHdb leads to a large array of secondary data (attributes): source of the allele (submitter, publication, or population); polymorphic haplotype background; and effect of the allele as predicted by molecular modeling on the phenylalanine hydroxylase enzyme (EC 1.14.16.1) or by in vitro expression analysis. The majority (63%) of the putative pathogenic PAH alleles are point mutations causing missense in translation of which few have a primary effect on PAH enzyme kinetics. Most apparently have a secondary effect on its function through misfolding, aggregation, and intracellular degradation of the protein. Some point mutations create new splice sites. A subset of primary PAH mutations that are tetrahydrobiopterin-responsive is highlighted on a Curators' Page. A clinical module describes the corresponding human clinical disorders (hyperphenylalaninemia [HPA] and phenylketonuria [PKU]), their inheritance, and their treatment. PAHdb contains data on the mouse gene (Pah) and on four orthologous mutant mouse models and their use (for example, in research on oral treatment of PKU with the enzyme phenylalanine ammonia lyase [EC 4.3.1.5]).     

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 ). Hum Mutat 11:4–17, 1998. © 1998 Wiley-Liss, Inc.     

The structural basis of phenylketonuria.

The human phenylalanine hydroxylase gene (PAH) (locus on human chromosome 12q24.1) contains the expressed nucleotide sequence which encodes the hepatic enzyme phenylalanine hydroxylase (PheOH). The PheOH enzyme hydroxylates the essential amino acid l-phenylalanine resulting in another amino acid, tyrosine. This is the major pathway for catabolizing dietary l-phenylalanine and accounts for approximately 75% of the disposal of this amino acid. The autosomal recessive disease phenylketonuria (PKU) is the result of a deficiency of PheOH enzymatic activity due to mutations in the PAH gene. Of the mutant alleles that cause hyperphenylalaninemia or PKU 99% map to the PAH gene. The remaining 1% maps to several genes that encode enzymes involved in the biosynthesis or regeneration of the cofactor ((6R)-l-erythro-5,6,7,8-tetrahydrobiopterin) regenerating the cofactor (tetrahydrobiopterin) necessary for the hydroxylation reaction. The recently solved crystal structures of human phenylalanine hydroxylase provide a structural scaffold for explaining the effects of some of the mutations in the PAH gene and suggest future biochemical studies that may increase our understanding of the PKU mutations.     

Characterization of phenylketonuria missense substitutions, distant from the phenylalanine hydroxylase active site, illustrates a paradigm for mechanism and potential modulation of phenotype

Missense mutations account for 48% of all reported human disease-causing alleles. Since few are predicted to ablate directly an enzyme's catalytic site or other functionally important amino acid residues, how do most missense mutations cause loss of function and lead to disease? The classic monogenic phenotype hyperphenylalaninemia (HPA), manifesting notably as phenylketonuria (PKU), where missense mutations in the PAH gene compose 60% of the alleles impairing phenylalanine hydroxylase (PAH) function, allows us to examine this question. Here we characterize four PKU-associated PAH mutations (F39L, K42I, L48S, I65T), each changing an amino acid distant from the enzyme active site. Using three complementary in vitro protein expression systems, and 3D-structural localization, we demonstrate a common mechanism. PAH protein folding is affected, causing altered oligomerization and accelerated proteolytic degradation, leading to reduced cellular levels of this cytosolic protein. Enzyme specific activity and kinetic properties are not adversely affected, implying that the only way these mutations reduce enzyme activity within cells in vivo is by producing structural changes which provoke the cell to destroy the aberrant protein. The F39L, L48S, and I65T PAH mutations were selected because each is associated with a spectrum of in vivo HPA among patients. Our in vitro data suggest that interindividual differences in cellular handling of the mutant, but active, PAH proteins will contribute to the observed variability of phenotypic severity. PKU thus supports a newly emerging paradigm both for mechanism whereby missense mutations cause genetic disease and for potential modulation of a disease phenotype.     

苯丙氨酸羟化酶基因突变及其与表型的关系研究进展

苯丙酮尿症(phenylketonuria,PKU)是一种可以造成儿童不同程度智力损害的常见的先天代谢性疾病,属于常染色体隐性遗传,OMIM编号为261600。PKU是由苯丙氨酸羟化酶(Phenylalanine hydroxylase,PAH)基因突变导致的肝脏PAH酶活性降低或丧失所致,1934年由Folling首先报道了此病。1983年该基因被定位在12q22-24.2。近些年,随着分子遗传学及生物学等科学的发展,对PKU研究也在不断深入。目前在国际PAH数据库(http//www.pahdb.mcgill.ca)中总共收录546种突变等位基因,659种基因型。PKU表型与基因突变类型有关,但是有些带有相同pah基因突变的不同PKU患者存在表型差异。全面了解pah基因结构、pah基因表达特点、酶结构和功能以及基因突变对酶结构和功能的影响的分子机制是对PKU表型认识的基础。本文根据近年来的研究成果,对pah基因结构,PAH酶结构,基因突变及其与表型的关系等进行详细阐述。     

In vitro expression and structural analysis of four missense mutations (G247S, E280G, P362T, A434D) of phenylalanine hydroxylase gene]

OBJECTIVE: To 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.     

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

PurposeThe 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.MethodsA total of 9336 PKU patients with 2589 different genotypes, carrying 588 variants, were investigated using an allelic phenotype value (APV) algorithm.ResultsWe 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).ConclusionsAPV and GPV are powerful tools to investigate genotype–phenotype associations, and can be used for genetic counselling of PKU families.     

Tetrahydrobiopterin,its Mode of Action on Phenylalanine Hydroxylase,and Importance of Genotypes for Pharmacological Therapy of Phenylketonuria

In about 20%–30% of phenylketonuria (PKU) patients (all phenotypes of PAH deficiency), Phe levels may be controlled through phenylalanine hydroxylase cofactor tetrahydrobiopterin therapy. These patients can be diagnosed by an oral tetrahydrobiopterin challenge and are characterized by mutations coding for proteins with substantial residual PAH activity. They can be treated with a commercially available synthetic form of tetrahydrobiopterin, either as a monotherapy or as adjunct to the diet. This review article summarizes molecular and metabolic bases of PKU and the importance of the tetrahydrobiopterin loading test used for PKU patients. On the basis of in vitro residual PAH activity, more than 1,200 genotypes from patients challenged with tetrahydrobiopterin were categorized as predictive for tetrahydrobiopterin responsiveness or non‐responsiveness and correlated with the loading test, phenotype, and residual in vitro PAH activity. The coexpression of two distinct PAH mutant alleles revealed possible dominance effects (positive or negative) by one of the mutations on residual activity as result of interallelic complementation. The treatment of the transfected cells with tetrahydrobiopterin showed an increase in residual PAH activity with several mutations coexpressed.     

The interplay between genotype, metabolic state and cofactor treatment governs phenylalanine hydroxylase function and drug response

The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH(4) cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH(4) concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH(4) loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH(4) ratio. Finally, evaluation of data from BH(4) loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH(4)-responsive PAH deficiency.     

Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria

Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) and increased levels of phenylalanine. PAH requires the cofactor BH(4) to function and the rate-limiting step in the synthesis of BH(4) is GTP cyclohydrolase I (GTP-CH). The skin is a potential target tissue for PKU gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin, to metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed a higher copy number of transgenes in keratinocytes and also a higher metabolic capacity.     

The V388M mutation results in a kinetic variant form of phenylalanine hydroxylase

The molecular mechanism underlying the metabolic defect in phenylketonuria (PKU) patients carrying the V388M missense mutation of the phenylalanine hydroxylase (PAH) gene has been characterized. An in vitro prokaryotic expression system has been used to produce both the wild-type and the mutant form of the human PAH (hPAH) protein. The recombinant enzymes, obtained as fusion proteins, were purified by immobilized metal affinity chromatography and recovered in high yields. The wild-type hPAH possessed a high specific activity and its kinetic properties were the same as those reported for the enzyme isolated from human liver and other recombinant wild-type hPAH enzymes. The recombinant V388M mutant form exhibited a reduced specific activity equivalent to 30% of the wild-type hPAH enzyme when assayed using the synthetic cofactor (6-methyltetrahydropterin). Lower values were obtained (23 and 19%) when the mutant enzyme was assayed with the natural cofactor ((6R)-tetrahydrobiopterin) and different concentrations of l-phenylalanine. The enzyme kinetic studies of the V388M mutant protein revealed that this enzyme was a kinetic variant form of hPAH with a reduced affinity for l-phenylalanine and for the natural cofactor ((6R)-tetrahydrobiopterin). The residual activities determined for the V388M form of hPAH were compatible with the phenotype presented by the PKU patients harboring the V388M mutation in the PAH gene.     

Molecular and phenotypic characteristics of patients with phenylketonuria in Serbia and Montenegro

Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Caucasians. PKU is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) enzyme. Here, we report the spectrum and the frequency of mutations in the PAH gene and discuss genotype-phenotype correlation in 34 unrelated patients with PKU from Serbia and Montenegro. Using both polymerase chain reaction-restriction fragment length polymorphism and 'broad-range' denaturing-gradient gel electrophoresis/DNA sequencing analysis, 19 disease-causing mutations were identified, corresponding to mutation detection rate of 97%. The most frequent ones were L48S (21%), R408W (18%), P281L (9%), E390G (7%) and R261Q (6%), accounting for 60% of all mutant alleles. The genotype-phenotype correlation was studied in homozygous and functionally hemizygous patients. We found that the most frequent mutation, L48S, was exclusively associated with the classical (severe) PKU phenotype. The mutation E390G gave rise to mild PKU. For the mutation R261Q, patients had been recorded in two phenotype categories. Considering allele frequencies, PKU in Serbia and Montenegro is heterogeneous, reflecting numerous migrations over the Balkan Peninsula.     

Toward mechanistic models for genotype–phenotype correlations in phenylketonuria using protein stability calculations

Phenylketonuria (PKU) is a genetic disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH), resulting in accumulation of phenylalanine to neurotoxic levels. Here, we analyzed the cellular stability, localization, and interaction with wild‐type PAH of 20 selected PKU‐linked PAH protein missense variants. Several were present at reduced levels in human cells, and the levels increased in the presence of a proteasome inhibitor, indicating that proteins are proteasome targets. We found that all the tested PAH variants retained their ability to associate with wild‐type PAH, and none formed aggregates, suggesting that they are only mildly destabilized in structure. In all cases, PAH variants were stabilized by the cofactor tetrahydrobiopterin (BH₄), a molecule known to alleviate symptoms in certain PKU patients. Biophysical calculations on all possible single‐site missense variants using the full‐length structure of PAH revealed a strong correlation between the predicted protein stability and the observed stability in cells. This observation rationalizes previously observed correlations between predicted loss of protein destabilization and disease severity, a correlation that we also observed using new calculations. We thus propose that many disease‐linked PAH variants are structurally destabilized, which in turn leads to proteasomal degradation and insufficient amounts of cellular PAH protein.     

Protective effect of recombinant adeno-associated virus 2/8-mediated gene therapy from the maternal hyperphenylalaninemia in offsprings of a mouse model of phenylketonuria

Phenylketonuria (PKU) is an autosomal recessively inherited metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and the fetus of an uncontrolled pregnant female patient presents with maternal PKU syndrome. We have reported previously on the cognitive outcome of biochemical and phenotypic reversal of PKU in a mouse model, Pahenu2, by the AAV serotype 2-mediated gene delivery of a human PAH transgene. However, the therapeutic efficacy had been limited to only male PKU mice. In this study, we generated a pseudotyped recombinant AAV2/8-hPAH vector and infused it into female PKU mice through the hepatic portal vein or tail vein. Two weeks after injection, complete fur color change to black was observed in female PKU, as in males. The PAH activity in the liver increased to 65-70% of the wild-type activity in female PKU mice and to 90% in male PKU mice. Plasma phenylalanine concentration in female PKU mice decreased to the normal value. In addition, the offsprings of the treated female PKU mice can rescue from the harmful effect of maternal hyperphenylalaninemia. These results indicate that recombinant AAV2/8-mediated gene therapy is a potential therapeutic strategy for PKU.     

Genotype and phenotype correlation of phenylalanine hydroxylase deficiency among patients from Henan北大核心CSCD

Objective: To delineate the mutation spectrum of phenylalanine hydroxylase (PAH) gene among patients affected with phenylalanine hydroxylase deficiency (PAHD) in Henan Province of China, and to explore the correlation between the genotype and the phenotype. Methods: A total of 155 affected children were recruited. Potential mutation of the PAH gene were analyzed by direct sequencing. The genotype - phenotype correlation was analyzed by matching the expected and observed phenotypes. Results: Over 72 mutations and 108 genotypes have been identified. There were 7 homozygous mutations, including 1 case with EX6-96A>G/EX6-96A>G, 1 with R241C/R241C, 1 with R413P/R413P, and 4 with R243Q/R243Q. Among these, 6 patients have presented classic PKU phenotypes, except for a R241C/R241C genotype which has led to mild PKU. In 104 patients carrying compound PAH mutations, 52 were classic, 34 were mild and 39 had mild HPA. Patients who were heterozygous for EX6-96A>G/R241C, R243Q/A434D, EX6-96A>G/R413P and EX6-96A>G/ R241C were found with both the classic PKU and mild PKU phenotypes. Common mutations associated with mild HPA have included R53H, R243Q, V399V and H107R. The common mutations associated with mild PKU included R243Q, R241C, EX6-96A>G, and IVS4-1G>A. The prevalent mutations in classic PKU were R243Q, EX6-96A>G and V399V. The consistency between prediction of the biochemical genotype and observed phenotype was 77.78%, especially in classic PKU, the consistency was up to 82.14%. Significant correlations were disclosed between pretreatment levels of phenylalanine and AV sum (r= -0.6729, P<0.01). Conclusion: The mutation spectrum of PAH gene in Henan seems to differ from that of other regions. Independent assortment of mutant alleles may result in a complex genotype-phenotype correlation, but the genotypes of PAHD patients have correlated with the phenotype. © 2016, West China University of Medical Sciences. All rights reserved.