Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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It has been postulated that the significant incidence of learning disabilities in well-treated patients with phenylketonuria (PKU) may be due, in part, to reduced production of neurotransmitters as a result of deficient tyrosine transport across the neuronal cell membrane. Hypotyrosinemia has been reported in treated and untreated PKU but virtually no data are available. We decided to examine this in our patient population and to compare it with the published norms, patient data from our hospital clinical biochemical laboratory database, and a group of normal children and adolescents in a private pediatric practice. We found that the mean nonfasting plasma tyrosine in 99 classical PKU patients was 41.1 micromol/L, in 26 mild (atypical) PKU patients 53.3 micromol/L, and in 35 non-PKU mild hyperphenylalaninemia patients 66.6 micromol/L. This compared to nonfasting plasma tyrosine levels in 102 non-PKU subjects of 64.0 micromol/L in our hospital biochemistry database, 69.1 micromol/L in 58 volunteers in the private office practice, and 64-78.8 micromol/L in infants, children, and adolescents in the literature review. Our data support the previously undocumented statements in the literature that plasma tyrosine levels are low in PKU.
Mol Genet Metab 2000 Apr
PMID:"Hypotyrosinemia" in phenylketonuria. 1087 Aug 46

Hyperphenylalaninemia, which can cause neurological disorders and mental retardation, results from a mutation in phenylalanine hydroxylase or an enzyme required for biosynthesis or regeneration of its cofactor, tetrahydrobiopterin. The hyperphenylalaninemia variant primapterinuria is characterized by the excretion of 7-biopterin (primapterin). This disorder is thought to be due to a deficiency of 4a-hydroxy-tetrahydrobiopterin dehydratase (pterin-4a-carbinolamine dehydratase), but a lack of tissue activity has not been directly demonstrated. The five mutations so far recognized in patients with primapterinuria are associated with either a single amino acid change or a premature stop codon. Only C81R has been successfully expressed in soluble form, and was found to have 40% of normal activity. Tissues which could be obtained by minimally invasive procedures were analyzed for dehydratase activity. None was detected in normal human white cells or fibroblasts. However, activity was found in intestine of rat, dog, pig, and particularly humans where it was only eight times lower than in liver. Distribution along the length and across the wall of small intestine was relatively uniform. Moreover, the dehydratases from human liver and intestinal mucosa have identical kinetic properties. A biopsy of duodenal mucosa from a patient with homozygous E96K dehydratase had activity of 55 nmol. min(-1)g(-1) mucosa compared to 329 +/- 32 nmol. min(-1)g(-1) tissue in controls (n = 12). The sixfold lower tissue activity of the E96K mutant alone may not be sufficient to account for the biochemical symptoms of primapterinuria in this patient. However, accumulation of a 4a-hydroxy-tetrahydrobiopterin degradation product (a side-chain cyclic adduct), which has been observed in vitro and appears to be a dehydratase inhibitor, may further exacerbate the problem.
Mol Genet Metab 2000 Jul
PMID:Hyperphenylalaninemia and 7-pterin excretion associated with mutations in 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH: analysis of enzyme activity in intestinal biopsies. 1092 72

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

Chemical chaperones are low molecular weight compounds known to stabilize proteins in vitro. Recently it was shown that, in transfected cells, these molecules can also correct the defective folding of some mutant proteins. Hyperphenylalaninemia (HPA) has been proposed to be classified as a "conformational disease," since it has been shown that the majority of the PAH mutations affect protein folding, thereby causing an increasing tendency toward aggregation and proteolytic degradation. Based on these observations, the effect of glycerol as a stabilizer agent of recombinant mutant forms of human phenylalanine hydroxylase enzymes (hPAH) produced in a prokaryotic expression system was investigated. The wild-type and two mutant forms of the hPAH protein (R270K and V388M) were expressed in the presence of glycerol in the culture medium. The yield, specific enzymatic activities, and kinetic properties of the recombinant proteins were determined and compared with the data obtained under normal growth conditions. The results obtained demonstrate that glycerol not only improved the yield of the soluble hPAH proteins (2- to 3-fold depending on the mutant enzyme) produced but also increased the specific activity of the purified recombinant enzymes. We speculate that correction of protein folding abnormalities by chemical chaperones may be a possible therapeutic approach to correct conformational diseases.
Mol Genet Metab 2001 Jun
PMID:Glycerol increases the yield and activity of human phenylalanine hydroxylase mutant enzymes produced in a prokaryotic expression system. 1138 53

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

DOPA responsive dystonia (DRD) and sepiapterin reductase (SR) deficiency are inherited disorders of tetrahydrobiopterin (BH4) metabolism characterized by the signs and symptoms related to monoamine neurotransmitter deficiency. In contrast to classical forms of BH4 deficiency DRD and SR deficiency present without hyperphenylalaninemia and thus cannot be detected by the neonatal screening for phenylketonuria (PKU). While DRD is mostly caused by autosomal dominant mutations in the GTP cyclohydrolase I gene (GCH1), SR deficiency is an autosomal recessive disease. The most important biochemical investigations for the diagnosis of these neurological diseases includes CSF investigations for neurotransmitter metabolites and pterins as well as neopterin and biopterin production in cytokine-stimulated fibroblasts. Discovery of SR deficiency opened new insights into alternative pathways of the cofactor BH4 via carbonyl, aldose, and dihydrofolate reductases. As a consequence of the low dihydrofolate reductase activity in the brain, dihydrobiopterin intermediate accumulates and inhibits tyrosine and tryptophan hydroxylases and uncouples nitric oxide synthase (nNOS), leading to neurotransmitter deficiency and possibly also to neuronal cell death.
Mol Genet Metab
PMID:Tetrahydrobiopterin deficiencies without hyperphenylalaninemia: diagnosis and genetics of dopa-responsive dystonia and sepiapterin reductase deficiency. 1159 14

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

Sepiapterin reductase (SR) deficiency was recently described in patients with a severe biogenic amine deficiency presenting without hyperphenylalaninemia and it was suggested that the tetrahydrobiopterin (BH(4)) pathway may be different in different cells and tissues. We now developed a HPLC method for the measurement of yellow fluorescing sepiapterin for the rapid diagnosis of SR deficiency. Sepiapterin was elevated in CSF from two patients with SR deficiency (5.6 and 11.4 nmol/L) when compared with healthy controls (<0.5 nmol/L). Our data further support the hypothesis that sepiapterin is an intermediate in the salvage pathway of BH(4) and that it accumulates in the brain of patients with SR deficiency.
Mol Genet Metab 2002 Feb
PMID:Detection of sepiapterin in CSF of patients with sepiapterin reductase deficiency. 1185 37


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