Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model

Treatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)—one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C>A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment.     

Predicting a clinical/biochemical phenotype for PKU/MHP patients with PAH gene mutations

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). In this study, a total of 218 independent PAH chromosomes (109 unrelated patients with PKU residing in Lithuania) were investigated. All 13 exons of the PAH gene of all PKU probands were scanned for DNA alterations by denaturing gradient gel electrophoresis (DGGE). In the cases of a specific DGGE pattern recognized, mutations were identified by direct fluorescent automated sequencing or by restriction enzyme digestion analysis of relevant exons. Twenty-five different PAH gene mutations were identified in Lithuania. We estimated a connection between individual PAH locus mutations and biochemical and metabolic phenotypes in patients in whom the mutant allele acts on its own, i.e., in functionally hemizygous patients and using the assigned value (AV) method to determine the severity of both common and rare mutant alleles, as well as to check a model to predict the combined phenotypic effect of two mutant PAH alleles. The text was submitted authors English.     

Phenylalanine ammonia-lyase modified with polyethylene glycol: Potential therapeutic agent for phenylketonuria

Summary. Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by the defects in the phenylalanine hydroxylase (PAH) gene. Individuals homozygous for defective PAH alleles show elevated levels of systemic phenylalanine and should be under strict dietary control to reduce the risk of neuronal damage associated with high levels of plasma phenylalanine. Researchers predict that plant phenylalanine ammonia-lyase (PAL), which converts phenylalanine to nontoxic t-cinnamic acid, will be an effective therapeutic enzyme for the treatment of PKU. The problems of this potential enzyme therapy have been the low stability in the circulation and the antigenicity of the plant enzyme. Recombinant PAL originated from parsley (Petroselinum crispum) chemically conjugated with activated PEG₂ [2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine] showed greatly enhanced stability in the circulation and was effective in reducing the plasma concentration of phenylalanine in the circulation of mice. PEG-PAL conjugate will be an effective therapeutic enzyme for the treatment of PKU.     

The PKU mutation S349P causes complete loss of catalytic activity in the recombinant phenylalanine hydroxylase enzyme

The mutation S349P in exon 10 of the phenylalanine hydroxylase (PAH) gene was identified in one Norwegian and one Polish phenylketonuria (PKU) allele on a haplotype 1.7 background. This missense mutation in PAH codon 349 is a T to C transition in cDNA position 1267. This mutation has been reported both on haplotype 1 and 4, suggesting recurrent mutation. In two different expression systems, the pET and the pMAL systems of Escherichia coli, it was shown that the S349P mutation, introduced by site directed mutagenesis, results in complete loss of enzymatic activity. Thus, protein instability alone does not seem to be the direct cause of the lack of activity of this PKU mutation as previously reported.We have identified mutations in the PAH gene of 118 PKU patients in Norway. To obtain information about how the different mutations affect the catalytic properties of the PAH enzyme we have used two prokaryotic expression systems.We detected the mutation S349P (Forrest et al. 1991) in one Norwegian patient and one of Polish ancestry. This mutation has previously been reported on haplotype 4 in North-African Jews (Weinstein et al. 1993), and on haplotype 1 in French-Canadians (John et al. 1992) and in Danes (Guldberg et al. 1993a). Here we present gene expression data showing that the recombinant mutant enzyme has no measurable residual catalytic activity.     

Phenylalanine hydroxylase deficiency in the Slovak population: Genotype–phenotype correlations and genotype-based predictions of BH4-responsiveness

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 135 Slovak PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed using high-resolution melting analysis, with subsequent sequencing analysis of the samples showing deviated melting profiles compared to control samples. The PAH gene was also screened for deletions and duplications using MLPA analysis. Forty-eight different disease causing mutations were identified in our patient group, including 30 missense, 8 splicing, 7 nonsense, 2 large deletions and 1 small deletion with frameshift; giving a detection rate of 97.6%. The most prevalent mutation was the p.R408W, occurring in 47% of all alleles, which concurs with results from neighboring and other Slavic countries. Other frequent mutations were: p.R158Q (5.3%), IVS12 + 1G>A (5.3%), p.R252W (5.1%), p.R261Q (3.9%) and p.A403V (3.6%). We also identified three novel missense mutations: p.F233I, p.R270I, p.F331S and one novel variant: c.− 30A>T in the proximal part of the PAH gene promoter. A spectrum of 84 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 36 were predicted to be BH4-responsive represented by 51 PKU families. In addition, genotype–phenotype correlations were performed.     

Molecular characterization of phenylketonuria in Japanese patients

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T278I (7.3%), E6nt-96A→g (6.1%), Y356X (4.9%), R111X (3.7%), and 442–706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83±0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99±0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9–1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder. Received: 24 July 1998 / Accepted: 12 September 1998     

Mutation spectrum of the <Emphasis Type="Italic">PAH</Emphasis> gene in phenylketonuria patients in the Karachay-Cherkess Republic (Russia)

A comprehensive population and medical-genetic study was carried out in ten districts and two cities in the Karachay-Cherkess Republic (Russia). As a result, 57 patients with phenylketonuria were revealed. PAH gene genotypes for 40 probands and their diseased and healthy relatives were determined. The mutation spectrum of the PAH gene in the Karachay-Cherkess Republic was investigated. The major mutation in this region is R261X with allelic frequency of 68.4%. We elaborated a convenient system for detection of six PAH gene mutations common in the Karachay-Cherkess Republic, with the total information content of the system being 89.9%. As a result of processing the clinical data, association of the diet and phenylalanine levels in the blood was verified. Genophenotypic analysis confirms the association of the residual activity of phenylalanine hydroxylase and the severity of the disease. It is shown that common mutation R261X is severe and that patients who are homozygous for this mutation have classical phenylketonuria (PKU).     

Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 Å from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity (k_cat) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The K_M values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3–4 in the Y179F mutant. However, the K_M values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the K_M value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of k_cat/K_Phe, the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AAAHs aromatic amino acid hydroxylases - BH₂ 7,8-dihydro-l-biopterin - BH₄ (6R)-5,6,7,8-tetrahydro-l-biopterin - CD circular dichroism - cPAH Chromobacterium violaceum phenylalanine hydroxylase - DMPH₄ 6,7-dimethyl-5,6,7,8-tetrahydropterin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - ES-MS electrospray ionization mass spectrometry - hPAH human phenylalanine hydroxylase - ICP-AE inductively coupled plasma atomic emission - 6-MPH₄ 6-methyl-5,6,7,8-tetrahydropterin - PAH phenylalanine hydroxylase - PH₄ tetrahydropterin - PKU phenylketonuria - RDS rate-determining step - TH tyrosine hydroxylase - THA 3-(2-thienyl)-l-alanine - TPH tryptophan hydroxylase - wt wild-type     

苯丙酮尿症分子遗传学研究进展

苯丙酮尿症是由于苯丙氨酸羟化酶基因突变引起的常染色体隐性遗传病。文章综述了苯丙酮尿症中的苯丙氨酸羟化酶基因的定位、结构、突变、调控以及突变基因的体外表达和苯丙氨酸羟化酶的三维结构特点等分子遗传学进展,阐述了苯丙氨酸羟化酶基因的突变对苯丙氨酸羟化酶的体外表达及其三维结构的影响, 以及部分基因型与表型相关的分子机制。 Abstract: Phenylketonuria(PKU) is one kinds of autusomal recessive disease caused by phenylalanine hydroxylase(PAH) gene mutation. This article reviews the recent molecular heredity progress on the phenylalanine hydroxylase gene’s orientation、structureand gene mutation and gene regulation. At same time, mutation gene in vitro expression and the character of 3D structure of PAH in PKU are involved. In this paper, also discussed the inflence of vitro expression and 3D protein structure by gene mutations and the molecular mechanism of the relationship between genotype and phenotype in PKU patient.     

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH^enu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH^enu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH^enu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH^enu2 mice were not protective against cancer.     

Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions

To simultaneously assess the effects of exonic mutations on RNA splicing and protein functions, we report here an intron-inclusive cDNA (Intinc) expression system. As a test model, twenty-four mutations in exon 9 of the phenylalanine hydroxylase (PAH) gene were examined in an Intinc expression plasmid composed of the PAH cDNA with the exon 9 flanked by its authentic introns. When the PAH enzyme activities from the Intinc plasmid-transfected cells were compared to those of a standard cDNA expression system, five mutations resulted in significant relative differences in PAH activities attributed to altered exon 9-inclusive mRNA levels. Two of the mutations affected exon recognition probably through splice site modifications and the remaining three affected experimentally verified exon splicing enhancer (ESE) motifs. The Intinc expression system allows not only a better link between mutation genotype to disease phenotype but also contributes to further understanding of molecular mechanisms of deleterious effects of mutations.     

Mutation spectrum of phenylketonuria in Syrian population: Genotype–phenotype correlation

Characterization of the molecular basis of phenylketonuria (PKU) in Syria has been accomplished through the analysis of 78 unrelated chromosomes from 39 Syrian patients with PKU. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by using molecular detection methods based on the restriction fragment length polymorphism (RFLP), artificial constructed restriction sites (ACRS) PCR and direct DNA sequencing. 56.4% of the patients had cPKU. A mutation detection rate of 79.49% was achieved and sixteen different mutations were found: missense 56.25%, splice site 37.5%, and frameshift 6.25%. The predominant mutation in this population sample was p.R261Q G>A, p.F55>Lfs and p.R243Q G>A. No mutation in six PKU patients was observed. In 57.9% of patient genotypes, the metabolic phenotype could be predicted. The identification of the mutations in the PAH gene and the genotype–phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counseling for the patient's relatives.     

Genotyping of patients with phenylketonuria from different regions of Russia for determining BH4 responsiveness

To date, the efficacy of the phenylalanine hydroxylase (PAH) cofactor is proved for the treatment of both BH4-dependent hyperphenylalaninemia and phenylketonuria patients with mutations in the PAH gene. Since the patient’s response depends on the presence of residual PAH enzyme activity, it is advisable to search for mutations in the PAH gene to identify the potential responders and nonresponders to therapy. Four hundred thirty-five phenylketonuria patients from 13 regions of the Russian Federation were genotyped in order to identify responders and nonresponders to tetrahydrobiopterin (BH4) therapy. According to the results of this study, the number of probable nonresponders to the BH4 treatment exceeds 50% owing to a higher overall allelic frequency of “severe” PAH gene mutations. Responder patients with two “mild” mutations in the PAH gene were identified (1.6%).     

A 3-base pair in-frame deletion of the phenylalanine hydroxylase gene results in a kinetic variant of phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive disease due to deficiency of a hepatic enzyme, phenylalanine hydroxylase (PAH). The absence of PAH activity results in typical PKU while persistence of a residual enzyme activity gives rise to variant forms of the disease. We report here a 3-base pair in-frame deletion of the PAH gene (delta 194) in a mild variant, with markedly reduced affinity of the enzyme for phenylalanine (Km = 160 nM), and we provide functional evidence for responsibility of the deletion in the mutant phenotype. Since the deletion was located in the third exon of the gene, which presents no homology with other hydroxylases, we suggest that exon 3 is involved in the specificity of the enzyme for phenylalanine. Finally, since none of the 98 PKU patients tested were found to carry this particular deletion, our study suggests that this molecular event probably occurred recently on the background of a haplotype 2 gene in Portugal.     

Two novel PAH gene mutations detected in Italian phenylketonuric patients

We report the identification by denaturing gradient gel electrophoresis and sequence analysis of two new phenylalanine hydroxylase (PAH) gene mutations (IVS4nt-2 and N207S) in single chromosomes of two unrelated Italian phenylketonuric (PKU) patients. Interestingly, mutation Y204C, found on the second mutant allele of family F1, has been previously detected in Chinese patients. Haplotype analysis showed that the latter mutation is linked to the same haplotype (H4) in both Chinese and Italian patients, suggesting a common origin. In vivo assessment of mutation severity indicates that N207S is associated with classic PKU. The identification of these two new mutations further extends the remarkable heterogeneity of the PAH locus in the Italian population. Received: 23 May 1996     

Phenylketonuria in Iranian population: a study in institutions for mentally retarded in Isfahan

Phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene (12q22-q24) resulting in a primary deficiency of the PAH enzyme activity, intolerance to the dietary intake of phenylalanine (Phe) and production of the phenylketonuria (PKU) disease. To date there have been no reports on the molecular analysis of PKU in Iranian population. In this study, the states of the PKU disease in terms of prevalence and mutation spectrum among patients reside in the institutions for mentally retarded in Isfahan was investigated. In the first step, 611 out of 1541 patients with PKU phenotype or severe mental retardation were screened for the PKU disease using the Guthrie bacterial inhibition assay (GBIA) followed by HPLC. Among the patients screened 34 (5.56%) were found positive with abnormal serum Phe of above 7mg/dl. In the next step, the presence of 18 common mutations of the PAH gene in 26 of the patients with classical PKU (serum Phe above 20mg/dl) was investigated, using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Of the 52 independent mutant alleles that were analyzed, 34 (65.38%) were genotyped showing 8 mutations as follows: R252W (15.38%), Q232Q (13.46%), R261Q (7.69%), delL364 (7.69%), IVS10-11g>a (5.77%), L333F (5.77%), V245V (5.77%) and S67P (3.85%). The results from this study may serve as a reference to analyze the PKU mutations in other part of Iran, and to establish diagnostic tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population.     

Mutational landscape of phenylketonuria in Iran

To date more than 1000 different variants in the PAH gene have been identified in patients with phenylketonuria (PKU). In Iran, several studies have been performed to investigate the genetics bases of the PKU in different parts of the country. In this study, we have analysed and present an update of the mutational landscape of the PAH gene as well as the population genetics and frequencies of detected variants for each cohort. Published articles on PKU mutations in Iran were identified through a comprehensive PubMed, Google Scholar, Web of Science (ISI), SCOPUS, Elsevier, Wiley Online Library and SID literature search using the terms: “phenylketonuria”, “hyperphenylalaninemia”, and “PKU” in combination with “Iran”, “Iranian population”, “mutation analysis”, and “Molecular genetics”. Among the literature-related to genetics of PKU, 18 studies were on the PKU mutations. According to these studies, in different populations of Iran 1497 patients were included for mutation detection that resulted in detection of 129 different mutations. Results of genetic analysis of the different cohorts of Iranian PKU patients show that the most prevalent mutation in Iran is the pathogenic splice variant c.1066-11G > A, occurring in 19.54% of alleles in the cohort. Four other common mutations were p.Arg261Gln, p.Pro281Leu, c.168 + 5G > C and p.Arg243Ter (8.18%, 6.45%, 5.88% and 3.7%, respectively). One notable feature of the studied populations is its high rate of consanguineous marriages. Considering this feature, determining the prevalent PKU mutations could be advantageous for designing screening and diagnostic panels in Iran.     

Activation of Phenylalanine Hydroxylase Induces Positive Cooperativity toward the Natural Cofactor

Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate l-Phenylalanine and its natural cofactor 6R-l-erythro-5,6,7,8-tetrahydrobiopterin (BH₄). Sapropterin dihydrochloride, the synthetic form of BH₄, was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH₄, a previously unknown finding. Analyses of structurally preactivated variants substantiated BH₄-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient''s metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.