Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phenylketonuria (PKU) is an inborn error of metabolism that is inherited in an autosomal recessive manner. It arises from a deficiency of phenylalanine hydroxylase, which is responsible for converting phenylalanine to tyrosine and thereby hastening its catabolism. To produce mouse models for the study of PKU, male mice were mutagenized with ethylnitrosourea and their progeny were screened for the elevated phenylalanine levels characteristic of phenylalanine hydroxylase deficiency. Of three mutant alleles recovered, two (Pah(enu1) and Pah(enu2)) were characterized previously and shown to be missense mutations. Sequencing of phenylalanine hydroxylase cDNA from the third mutant allele, Pah(enu3), revealed that two differently sized transcripts were being produced. These transcripts contained either a 5-nucleotide insertion or a 5-nucleotide deletion and both of these modifications occurred at the same location, the exon 11-exon 12 junction. Sequencing of the exon 11-intron 11 boundary revealed a T --> G transversion in the invariant GT dinucleotide of the wild-type 5' splice donor site. The analogous human Pah mutation would be called c.1199 + 2T > G. Sequence analysis also revealed two cryptic splice donor sites, upstream and downstream of the wild-type splice site, that appear to be used when the wild type is ablated and to thereby yield the observed differently sized transcripts. The 5-nucleotide insertion and the 5-nucleotide deletion are both predicted to cause frame shifting in exon 12 and exon 13, leading to premature termination.
Mol Genet Metab 2001 Jan
PMID:Characterization of the mouse phenylalanine hydroxylase mutation Pah(enu3). 1116 25

Phenylalanine hydroxylase (PAH) is a homotetrameric enzyme that catalyzes the conversion of phenylalanine to tyrosine, the rate-limiting step of phenylalanine disposal in humans. Primary dysfunction of PAH caused by mutations in the PAH gene results in hyperphenylalaninemia, which may impair cognitive development unless corrected by dietary restriction of phenylalanine. The mechanism(s) by which PAH missense mutations cause enzyme impairment has been studied in detail only in a small number of cases, but existing evidence points to a major role of enhanced proteolytic degradation due to aberrant folding of mutant polypeptides. We have used two heterologous in vitro expression systems (a mammalian cell-free transcription-translation system and the pET system of Escherichia coli) to examine 34 mutations that have been associated with PAH deficiency in the Danish population. These mutations represent a broad range of amino acid substitutions, functional enzyme domains, and metabolic phenotypes. In both systems, residual in vitro activities correlated broadly with metabolic phenotypes, however, with significant discrepancies. Analysis of E. coli extracts by nondenaturing polyacrylamide gel electrophoresis and storage experiments showed that (i) in general, mutations in the N-terminal regulatory domain are associated with relatively stable proteins compared to most mutations in the central catalytic domain, and (ii) for mutations in the catalytic domain, high levels of protein aggregation do not always correspond with a severe phenotype. Our data support and extend previous evidence that PAH mutations exert their pathogenic effects by several distinct mechanisms that may operate individually or in concert.
Mol Genet Metab 2001 Feb
PMID:In vitro expression of 34 naturally occurring mutant variants of phenylalanine hydroxylase: correlation with metabolic phenotypes and susceptibility toward protein aggregation. 1116 39

The elucidation of the molecular basis of hyperphenylalaninemia in various world populations (PKU Consortium Database: http://www.mcgill/ca/pahdb/) has revealed a remarkable molecular heterogeneity at the locus encoding for phenylalanine hydroxylase. As a consequence, genotyping of HPA patients has prompted the establishment of an impressive number of mutatIon detection protocols. In spite of the large variety of methods proposed so far, no comprehensive strategy has been yet developed for the detection of PAH gene mutations. Therefore, new approaches, combining the advantages of individual methods are required, especially in populations with a high number of PAH gene mutations. In this study, we propose the use of Reverse Dot Blot Analysis within a general mutation protocol to simplify the genotyping of hyperphenylalaninemics in the very heterogeneous population of Sicily (Italy).
Mol Cell Probes 2001 Feb
PMID:A methodological strategy for PAH genotyping in populations with a marked molecular heterogeneity of hyperphenylalaninemia. 1128 32

Recently, BH(4)-responsive phenylalanine hydroxylase (PAH) deficiency was reported in patients with specific mutations in the PAH gene, and it was suggested that BH(4) responsiveness may be determined by the respective genotypes. We now report on three patients with PAH deficiency and the same genotype but different responses to standardized BH(4) loading. Our results suggest that BH(4) responsiveness in PAH deficiency is at least partly independent from PAH genotype.
Mol Genet Metab 2001 May
PMID:Tetrahydrobiopterin responsiveness in phenylketonuria differs between patients with the same genotype. 1135 Jan 90

Phenylketonuria is one of the most common genetic diseases in humans, affecting 1 in 10,000 whites. Deletions are generally uncommon in genes in which no long highly homologous segments are present, and in phenylalanine hydroxylase (PAH) deficiency they represent only 5% of cases. We present the case of a girl affected by classical phenylketonuria who has been screened for mutations in the PAH gene. During the molecular study a large de novo deletion has detected in 12qter, including PAH, and the genes for insulin-like growth factor 1 (IGF1), human achaete-scute homolog 1 (ASCL1), and tumor rejection antigen (TRA1). The patient showed phenylketonuria, short stature, and pathological electro-oculography results in both eyes, with high affectation of the relative electrogenesis of the photoreceptor-pigment epithelium complex. She had previously been misdiagnosed as homozygous for the IVS8nt-7A-G mutation, instead of heterozygous for a mutation and a de novo deletion. As a result incorrect genetic counseling had been given. The deletion of the PAH, IGF1, and ASCL1 genes could explain the patient's phenotype corresponding to a contiguous gene syndrome. We stress the relevance of polymorphic marker haplotype analysis and the importance of family study in genetic recessive diseases, such as phenylketonuria, to avoid incorrect diagnosis and genetic counseling.
J Mol Med (Berl) 2001
PMID:Large de novo deletion in chromosome 12 affecting the PAH, IGF1, ASCL1, and TRA1 genes. 1143 25

Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase gene (PAH), while mutations in genes encoding the two enzymes (dihydropteridine reductase, DHPR, and pterin-4-alpha-carbinolamine dehydratase, PCD) required for recycling of its cofactor, tetrahydrobiopterin (BH(4)), cause other rarer disease forms of hyperphenylalaninemia. We have applied a yeast two-hybrid method, in which protein--protein interactions are measured by four reporter gene constructs, to the analysis of six PKU-associated PAH missense mutations (F39L, K42I, L48S, I65T, A104D, and R157N). By studying homomeric interactions between mutant PAH subunits, we show that this system is capable of detecting quite subtle aberrations in PAH oligomerization caused by missense mutations and that the observed results generally correlate with the severity of the mutation as determined by other expression systems. The mutant PAH subunits are also shown in this system to be able to interact with wild-type PAH subunits, pointing to an explanation for apparent dominant negative effects previously observed in obligate heterozygotes for PKU mutations. Based on our findings, the applications and limitations of two-hybrid approaches in understanding mechanisms by which PAH missense mutations exert their pathogenic effects are discussed. We have also used this technique to demonstrate homomeric interactions between wild-type DHPR subunits and between wild-type PCD subunits. These data provide a basis for functional studies on HPA-associated mutations affecting these enzymes.
Mol Genet Metab 2001 Jul
PMID:Homomeric and heteromeric interactions between wild-type and mutant phenylalanine hydroxylase subunits: evaluation of two-hybrid approaches for functional analysis of mutations causing hyperphenylalaninemia. 1146 Nov 90

Mutations in the gene encoding phenylalanine hydroxylase (PAH, EC 1.14.16.1) are associated with various degrees of hyperphenylalaninemia, including classical phenylketonuria (PKU). We examined the PAH gene in a Brazilian PKU family of African origin and identified three missense variants, R252W (c.754C --> T), K274E (c.820A --> G), and I318T (c.953T --> C), the two latter of which were transmitted in cis. Expression analyses in two different in vitro systems showed that I318T is associated with profoundly decreased enzyme activity, whereas the enzyme activity of K274E is indistinguishable from that of the wild-type protein. Detailed kinetic analyses of PAH expressed in E. coli showed that the K274E mutant protein has kinetic properties similar to that of the wild-type protein. Population studies have suggested that the K274E variant occurs on approximately 4% of African-American PAH alleles, whereas the neonatal screening incidence of PKU among African Americans is only 1:100,000. This is to our knowledge the first demonstration of a PAH missense variant with no apparent association to PAH deficiency. Awareness of this common variant may be helpful to laboratories that perform molecular diagnosis of PAH deficiency in populations of African origin.
Mol Genet Metab 2001 Jul
PMID:A phenylalanine hydroxylase amino acid polymorphism with implications for molecular diagnostics. 1146 Nov 96

The molecular basis of PAH deficiency in the Sicilian population is characterized by a marked heterogeneity, with 44 mutations at a single locus identified by a "gene-scanning" approach and accounting for a detection rate of 91%. The remaining 9% of PAH alleles does not bear mutations in any of the 13 exons and 24 exon/intron junctions. Three mutations IVS10nt-11 G > A, R261Q, and A300S accounted for 30.5%, whereas the remaining mutations were found at relative frequencies of less than 5% and 20 mutations were observed once only. Five mutations have been detected only in Sicilians so far. By studying the association of mutations with intragenic STR-VNTR haplotypes ("minihaplotypes"), "identity by descent" has been established for 24 mutations also detected in other populations. This finding supports the hypothesis of a multipolar origin for a large proportion of PAH mutant alleles currently detected in Sicilians. In order to improve our understanding of the clinical heterogeneity of PAH deficiency in this population, we have for the first time analyzed three missense mutations L41F, T92I, and P211T in vitro by the pCDNA3/COS-7 eukaryotic expression system and found an activity of 10, 76, and 72%, respectively, compared to normal PAH. In two HPA patients with mild PKU and mild hyperphenylalaninemia (MHP), harboring respectively L41F/R261Q and T92I/P281L genotypes, the predicted biochemical effect of these genotypes appeared to be consistent with the metabolic phenotypes. In contrast, discordant metabolic phenotypes (mild PKU and MHP) were observed in two unrelated patients bearing the same R261Q/P211T genotype, a finding which underscores the complex relationship linking genotype to phenotype in PAH deficiency. Hypotheses on the possible mechanisms responsible for the observed discordance are discussed. The spectrum of PAH gene mutations in Sicily reflects the complex demographic history of this island at the crossroad of prehistoric and historical migrations in the Mediterranean sea. The data presented in this study also add to the present knowledge on the relationship between PAH genotypes and HPA phenotype and are expected to improve PAH genotyping among individuals with hyperphenylalaninemia.
Mol Genet Metab 2001 Nov
PMID:PAH gene mutations in the Sicilian population: association with minihaplotypes and expression analysis. 1170 66

Several reports indicate that biopterin and folate pathways may interact. We examined folate metabolism in PKU patients where hyperphenylalaninaemia leads to a likely excess of THB. We found an increase in total HPLC determined red cell folate in PKU (p=0.0422): specifically, there was an increase in total formyl-H(4)folate (p=0.0002) and H(4)folate (p< or =0.0001), and decrease in total 5-methyl-H(4)folate in PKU patients. At the level of individual oligo-gamma-glutamyl coenzymes, we found that formyl-H(4)folate polyglutamates were virtually all increased in PKU (p=0.0223, 0.0004, 0.0004, 0.0012, and 0.0008 for di-, tri-, tetra-, penta-, and deca-gamma-glutamyl formyl-H(4)folate coenzymes, respectively). Hcy levels did not differ between clinical groups, indicating that folate dependent-Hcy remethylation is not compromised as a consequence of an altered PKU folate disposition. In nature, pentaglutamyl folates are considered the metabolically favoured coenzymes (optimum K(m) for dependent enzymes). The presented data support this-we found that red cell pentaglutamates gave the best measure of metabolism; pentaglutamyl formyl-H(4)folate increased in PKU (p=0.0012) and related methenyls behaved similarly, while, pentaglutamyl 5-methyl-H(4)folate and pentaglutamyl H(4)folate decreased (p< or =0.0001 and 0.0265, respectively). Furthermore, pentaglutamates showed the best correlations between one-carbon oxidation states of folate, as well as with Hcy (p=0.0003 r=-0.54, 95% CI; -0.724 to -0.272). That PKU might influence folate metabolism in some way is unsurprising: patients with DHPR deficiency accumulate DHB and develop secondary folate deficiency-responsive only to reduced folates, while CSF levels of THB are significantly correlated to monoamines and red cell folate in depression. Further studies to confirm the present findings and to ascertain precisely what mechanism operates in PKU that impacts upon folate homeostasis so profoundly are required.
Mol Genet Metab 2002 Aug
PMID:The impact of phenylketonuria on folate metabolism. 1220 35

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.
Mol Genet Metab 2002 Aug
PMID:Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria. 1220 36


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