The Pah-R261Q mouse reveals oxidative stress associated with amyloid-like hepatic aggregation of mutant phenylalanine hydroxylase.

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.

Molecular characterization of Thai patients with phenylalanine hydroxylase deficiency and in vitro functional study of two novel PAH variants.

Phenylketonuria (PKU) is an autosomal recessive amino acid metabolism disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH; EC1.14.16.1). This study aimed to assess the specific heterogeneity of PAH variants found in Thai population as well as evaluate enzyme activity and expression of novel variants. PAH gene from 13 patients was analyzed by PCR amplification and direct Sanger-sequencing of 13 exons of the coding region. The novel variants were transiently transfected in COS-7 cells for functional verification. Eleven different PAH variants were identified: all pathogenic variants were missense variants, of which the most frequent variant was p.R169L, accounting for 24% (6/25) of all identified alleles. Two novel variants p.R169L and p.Y317N and previously reported variants with mutated residues at the same positions (p.R169H and p.Y317H) were expressed in COS-7 cells. These showed mildly impaired residual activity levels (42.3-63.1% of wild type), while the protein levels were well expressed (82.8-110%), except for p.R169L, which showed decreased protein expression of 55.7% compared to the wild type enzyme. All subjects with p.R169L identified in at least one of pathogenic alleles (one case is homozygous) had a metabolic phenotype of mild hyperphenylalaninemia (HPA). Our data has expanded the information on the genetic heterogeneity of Thai patients with PAH deficiency. This finding emphasizes the importance of genotyping in patients with HPA, and in vitro studies can provide additional information for prediction of phenotype.

GTP-cyclohydrolase deficiency induced peripheral and deep microcirculation dysfunction with age.

The present study assessed the impact of impaired tetrahydrobiopterin (BH4) production on vasoreactivity from conduit and small arteries along the vascular tree as seen during aging. For this purpose, the mutant hyperphenylalaninemic mouse (hph-1) was used. This model is reported to be deficient in GTP cyclohydrolase I, a rate limiting enzyme in BH4 biosynthesis. BH4 is a key regulator of vascular homeostasis by regulating the nitric oxide synthase 3 (NOS3) activity. In GTP-CH deficient mice, the aortic BH4 levels were decreased, by -77% in 12 week-middle-aged mice (young) and by -83% in 35-45 week-middle-aged mice (middle-aged). In young hph-1, the mesenteric artery ability to respond to flow was slightly reduced by 9%. Aging induced huge modification in many vascular functions. In middle-aged hph-1, we observed a decrease in aortic cGMP levels, biomarker of NO availability (-46%), in flow-mediated vasodilation of mesenteric artery (-31%), in coronary hyperemia response measured in isolated heart following transient ischemia (-27%) and in cutaneous microcirculation dilation in response to acetylcholine assessed in vivo by laser-doppler technic (-69%). In parallel, the endothelium-dependent relaxation in response to acetylcholine in conduit blood vessel, measured on isolated aorta rings, was unchanged in hph-1 mice whatever the age. Our findings demonstrate that in middle-aged GTP-CH depleted mice, the reduction of BH4 was characterized by an alteration of microcirculation dilatory properties observed in various parts of the vascular tree. Large conduit blood vessels vasoreactivity, ie aorta, was unaltered even in middle-aged mice emphasizing the main BH4-deletion impact on the microcirculation.

Manipulation of a cation-π sandwich reveals conformational flexibility in phenylalanine hydroxylase.

Phenylalanine hydroxylase (PAH) is an allosteric enzyme that maintains phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria, an inborn error of amino acid metabolism. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich involving Phe80 that cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared and evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change. Limited proteolysis of WT rPAH (minus Phe) reveals facile cleavage within a 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of that tetramer. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH interchange. Hypersensitivity is corrected by addition of Phe such that all variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing sampling of on-pathway intermediates in the RS-PAH and A-PAH interchange. The behavior of the Phe80 variants mimics that of disease-associated R68S and suggests a molecular basis for proteolytic susceptibility in PKU-associated human PAH variants.

Pegvaliase for the treatment of phenylketonuria: Results of the phase 2 dose-finding studies with long-term follow-up.

BACKGROUND: Phenylketonuria (PKU) is characterized by a deficiency in phenylalanine hydroxylase (PAH) that may lead to elevated blood phenylalanine (Phe) and significant neurocognitive and neuropsychological comorbidities. Pegvaliase (PALYNZIQ®, BioMarin Pharmaceutical Inc.) is a PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL), which converts Phe to trans-cinnamic acid and ammonia, and was approved in May 2018 in the United States and in May 2019 in the European Union for decreasing blood Phe levels in adults with PKU with blood Phe levels >600 µmol/L. The efficacy and safety of pegvaliase was assessed in two phase 2 dose-finding studies in adults with PKU (PAL-002, NCT00925054, and PAL-004, NCT01212744). Participants completing these studies could enroll in a long-term extension study (PAL-003, NCT00924703). METHODS: Participants in PAL-002 received pegvaliase 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg weekly for 8 weeks, then continued treatment for a further 8 weeks with dose and/or frequency adjusted to achieve blood Phe concentrations of 60 to 600 µmol/L. Participants in PAL-004 received pegvaliase 0.001 to 0.4 mg/kg 5 days/week for 13 weeks, with modifications made to the starting dose in response to safety and/or efficacy, followed by 3 additional weeks of follow-up assessments. The maximum allowable daily dose in both studies was 1.0 mg/kg/day (5.0 mg/kg/week). Participants who completed any of the phase 2 studies (PAL-002; PAL-004; or a third phase 2 study, 165-205) were eligible to enroll in an open-label, multicenter, long-term extension study (PAL-003, NCT00924703). RESULTS: Thirty-seven of the 40 enrolled participants completed PAL-002 and 15 of the 16 enrolled participants completed PAL-004. Mean blood Phe at baseline was 1311.0 (standard deviation [SD] 354) µmol/L in PAL-002 and 1482.1 (SD 363.5) µmol/L in PAL-004. Mean blood Phe did not substantially decrease with pegvaliase treatment in PAL-002 (-206.3 [SD 287.1] µmol/L at Week 16) or PAL-004 (-410.8 [SD 653.7] µmol/L at Week 13). In PAL-004, mean blood Phe dropped from baseline by 929.1 µmol/L (SD 691.1) by Week 2; subsequent to dose modifications and interruptions, this early decrease in mean Phe level was not sustained. With increased pegvaliase dose and duration in PAL-003, mean blood Phe levels steadily decreased from baseline, with mean reductions by Week 120 of 68.8% (SD 44.2%) in PAL-002 participants and 75.9% (SD 32.4%) in PAL-004 participants. All participants in PAL-002 and PAL-004 reported ≥1 adverse event (AE), with higher exposure-adjusted event rates in PAL-004. The majority of AEs were mild (87.2% in PAL-002, 86.7% in PAL-004) or moderate (12.4% in PAL-002, 13.3% in PAL-004). The most commonly reported AEs in PAL-002 were injection site reaction (50.0% of participants), headache (42.1%), injection site erythema (36.8%), nausea (34.2%), and arthralgia (29.0%), and in PAL-004 were arthralgia (75.0%), headache (62.5%), dizziness (56.3%), injection site erythema (56.3%), and injection site reaction (50.0%). CONCLUSIONS: In two phase 2 dose-finding studies, pegvaliase did not lead to substantial blood Phe reductions. Higher and more frequent pegvaliase dosing in PAL-004 led to a substantial initial drop in blood Phe, but an increase in the number of hypersensitivity AEs and dose reductions or interruptions. With increased dose and duration of treatment in PAL-003, mean blood Phe reduction was substantial and sustained, and the frequency of hypersensitivity AEs decreased and stabilized. Together, these studies led to the development of an induction-titration-maintenance regimen that has been approved for pegvaliase, with patients starting at a low weekly dose that gradually increases in dose and frequency until they achieve a standard non-weight-based daily maintenance dose. This regimen has been tested in a third phase 2 study, as well as in two successful phase 3 studies of pegvaliase.

A microparticulate based formulation to protect therapeutic enzymes from proteolytic digestion: phenylalanine ammonia lyase as case study.

Phenylketonuria is a genetic disorder affecting the metabolism of phenylalanine (phe) due to a deficiency in the enzyme phenylalanine hydroxylase. This disorder is characterized by an elevated phe blood level, which can lead to severe intellectual disabilities in newborns. The current strategy to prevent these devastating consequences is limited to a life-long phe-free diet, which implies major lifestyle changes and restrictions. Recently, an injectable enzyme replacement therapy, Pegvaliase, has been approved for treating phenylketonuria, but is associated with significant side-effects. In this study a phe-metabolizing system suitable for oral delivery is designed to overcome the need for daily injections. Active phenylalanine ammonia-lyase (PAL), an enzyme that catalyses phe metabolism, is loaded into mesoporous silica microparticles (MSP) with pore sizes ranging from 10 to 35 nm. The surface of the MSP is lined with a semipermeable barrier to allow permeation of phe while blocking digestive enzymes that degrade PAL. The enzymatic activity can be partially preserved in vitro by coating the MSP with poly(allylamine) and poly(acrylic acid)-bowman birk (protease inhibitor) conjugate. The carrier system presented herein may provide a general approach to overcome gastro-intestinal proteolytic digestion and to deliver active enzymes to the intestinal lumen for prolonged local action.

Outcomes in pediatric studies of medium-chain acyl-coA dehydrogenase (MCAD) deficiency and phenylketonuria (PKU): a review.

BACKGROUND: Inherited metabolic diseases (IMDs) are a group of individually rare single-gene diseases. For many IMDs, there is a paucity of high-quality evidence that evaluates the effectiveness of clinical interventions. Clinical effectiveness trials of IMD interventions could be supported through the development of core outcome sets (COSs), a recommended minimum set of standardized, high-quality outcomes and associated outcome measurement instruments to be incorporated by all trials in an area of study. We began the process of establishing pediatric COSs for two IMDs, medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and phenylketonuria (PKU), by reviewing published literature to describe outcomes reported by authors, identify heterogeneity in outcomes across studies, and assemble a candidate list of outcomes. METHODS: We used a comprehensive search strategy to identify primary studies and guidelines relevant to children with MCAD deficiency and PKU, extracting study characteristics and outcome information from eligible studies including outcome measurement instruments for select outcomes. Informed by an established framework and a previously published pediatric COS, outcomes were grouped into five, mutually-exclusive, a priori core areas: growth and development, life impact, pathophysiological manifestations, resource use, and death. RESULTS: For MCAD deficiency, we identified 83 outcomes from 52 articles. The most frequently represented core area was pathophysiological manifestations, with 33 outcomes reported in 29/52 articles (56%). Death was the most frequently reported outcome. One-third of outcomes were reported by a single study. The most diversely measured outcome was cognition and intelligence/IQ for which eight unique measurement instruments were reported among 14 articles. For PKU, we identified 97 outcomes from 343 articles. The most frequently represented core area was pathophysiological manifestations with 31 outcomes reported in 281/343 articles (82%). Phenylalanine concentration was the most frequently reported outcome. Sixteen percent of outcomes were reported by a single study. Similar to MCAD deficiency, the most diversely measured PKU outcome was cognition and intelligence/IQ with 39 different instruments reported among 82 articles. CONCLUSIONS: Heterogeneity of reported outcomes and outcome measurement instruments across published studies for both MCAD deficiency and PKU highlights the need for COSs for these diseases, to promote the use of meaningful outcomes and facilitate comparisons across studies.

State-of-the-Art 2019 on Gene Therapy for Phenylketonuria.

Phenylketonuria (PKU) is considered to be a paradigm for a monogenic metabolic disorder but was never thought to be a primary application for human gene therapy due to established alternative treatment. However, somewhat unanticipated improvement in neuropsychiatric outcome upon long-term treatment of adults with PKU with enzyme substitution therapy might slowly change this assumption. In parallel, PKU was for a long time considered to be an excellent test system for experimental gene therapy of a Mendelian autosomal recessive defect of the liver due to an outstanding mouse model and the easy to analyze and well-defined therapeutic end point, that is, blood l-phenylalanine concentration. Lifelong treatment by targeting the mouse liver (or skeletal muscle) was achieved using different approaches, including (1) recombinant adeno-associated viral (rAAV) or nonviral naked DNA vector-based gene addition, (2) genome editing using base editors delivered by rAAV vectors, and (3) by delivering rAAVs for promoter-less insertion of the PAH-cDNA into the Pah locus. In this article we summarize the gene therapeutic attempts of correcting a mouse model for PKU and discuss the future implications for human gene therapy.

Transient phenylketonuria in premature infants.

Phenylketonuria (PKU) is an autosomal recessive inborn error of phenylalanine (phe) metabolism caused by a deficiency in the enzyme phenylalanine hydroxylase that converts phe into tyrosine. If left untreated, PKU results in increased phe concentrations in the blood and in the brain, which cause severe intellectual disability, epilepsy, and behavioral problems. These disorders can be prevented if a diet low in phe is introduced. This report focuses on a preterm newborn (gestational age 29 wk) with adequate weight (1290 g) and no family history of PKU. His parents had not received metabolic neonatal screening. A blood sample at 16 d of age and a weight of 1430 g showed phe 420 µmol/L, compatible with mild PKU. Mixed feeding was initiated with a formula free of phe (X-Phe), and breastfeeding was fortified with a contribution of 3.5 g/kg daily (2.5 g X-Phe and 1 g of high-value biological proteins). The next measurements of amino acid levels in the blood and urine were normal, and the progenitors study for PKU was negative. Normal feeding was reintroduced with normal neurologic and metabolic later evolution. The disorders of the metabolism of phe, in most cases, are due to a genetic condition. However, there are infrequent cases of transient hyperphenylalaninemia secondary to delayed maturation of the hydroxylation enzyme system. They are especially significant in premature infants. Although these forms have not been shown to cause sequelae, in view of high levels of phe in the blood, phe consumption must be restricted.

Relationship between genotype, phenylalanine hydroxylase expression and in vitro activity and metabolic phenotype in phenylketonuria.

Residual phenylalanine hydroxylase (PAH) activity is the main determinant of the metabolic phenotype in phenylketonuria (PKU). The genotypic heterogeneity of PKU, involving >1000 PAH variants and over 2500 different genotypes, makes genotype-based phenotype prediction challenging. While a relationship between PAH variants and the metabolic phenotype is well established, we questioned the importance of PAH expression and residual in vitro activity for the metabolic phenotype. Thirty-four PAH variants (p.F39 L, p.A47V, p.D59Y, p.I65S, p.R68G, p.R68S, p.E76G, p.A104D, p.D143G, p.R155H, p.R176L, p.V190A, p.G218 V, p.R241C, p.R243Q, p.P244L, p.R252W, p.R261Q, p.E280K, p.R297H, p.A300S, p.I306V, p.A309V, p.L311P, p.A313T, p.L348 V, p.V388 M, A403V, p.R408Q, p.R408W, p.R413P, p.D415N, p.Y417H, and p.A434D) were transiently transfected into COS-7 cells, and expression of PAH was investigated. Expression patterns were compared with in vitro PAH activity and allelic phenotype values (APVs). In vitro PAH activity was significantly higher (p < .01) in variants associated with mild hyperphenylalaninemia (PAH activity = 52.1 ±â€¯8.5%; APV = 6.7-10.0) than that in classic PKU variants (PAH activity = 21.1 ±â€¯7.0%; APV = 0-2.7). Mild PKU variants (PAH activity = 40.2 ±â€¯7.6%; APV = 2.8-6.6) were not significantly different from mild hyperphenylalaninemia, but there was a difference (p < .048) compared with classic PKU phenotypes.

Mutational spectrum of the phenylalanine hydroxylase gene in patients with phenylketonuria in the central region of China.

Phenylketonuria (PKU, OMIM 261600) caused by phenylalanine hydroxylase (PAH) deficiency is an autosomal recessive disease that is characterized by abnormalities of phenylalanine metabolism. In this study, a total of 77 patients, originating from the central region of China and who were diagnosed with PAH deficiency at the third affiliated hospital of Zhengzhou University, were enrolled in this study. The 13 exons and 12 flanking introns of the PAH gene were analyzed by Sanger sequencing and next generation sequencing. The sequencing data were aligned to the hg19, PAHvdb and HGMD databases to characterize the genotypes of PKU patients, and genotype-phenotype correlations and BH4 responsiveness predictions were performed using BIOPKUdb. In total, 149 alleles were characterized among the 154 PKU alleles. These mutations were located in exons 2-13, and intron 12 of the PAH gene, with a relative frequency of ≥5%, for EX6-96A>G, p.R241C, p.R243Q, p.V399V and p.R53H. Additionally, a novel variant, p.D84G, was identified. The genotype correlated with clinical symptoms in 33.3-100% of the cases, depending on the disease severity, and BH4 responsiveness predictions show that only five patients with MHP-PKU and one patient with Mild-PKU were predicted to be BH4 responsive. In conclusion, we have characterized the mutational spectrum of PAH in the central region of China and have identified a novel mutation. The hotspot mutation information might be useful for screening, diagnosis and treatment of PKU.

PAH mutation spectrum and correlation with PKU manifestation in north Jiangsu province population.

Phenylketonuria (PKU) is a common autosomal recessive disorder of phenylalanine metabolism and mainly results a deficiency of phenylalanine hydroxylase gene (PAH). The incidence of various PAH mutations have race and ethnicity differences. We report a spectrum of PAH mutations complied from 35 PKU children who are all Chinese Han population from north Jiangsu in this study. All 13 exons and their flanking intron sequences of PAH were determined by Ion Torrent PGM™ sequencing. The relationship of genotype and phenotype was analyzed based on the sum of the arbitrary value (AV) values of the two alleles. We identified 61 mutations, with a frequency of 87.14%, among 70 alleles of 35 patients. The most prevalent mutations were R243Q (26.23%), R241C (9.84%) and V399V (8.20%). Furthermore, the consistency between prediction of the biochemical phenotype and the observed phenotype was 81.25%, with the highest consistency observed in classic PKU (87.50%). A significant correlation was found between pretreatment levels of phenylalanine and AV sum (r = -0.87, P < 0.05). Finally, our study constructs PAH mutation spectrum by next generation sequencing (NGS), and reveals that the PAH genotypes and biochemical phenotypes were significantly correlated. These offers facilitate the provision of appropriate genetic counseling for PKU patients.

[Characteristics of phenylalanine hydroxylase gene mutations among patients with phenylketonuria from Linyi region of Shandong Province].

OBJECTIVE: To explore the characteristics of (PAH) gene mutations among patients with phenylketonuria (PKU) from Linyi area of Shandong Province. METHODS: For 51 children affected with PKU and their parents, the 13 exons and their flanking intronic sequences of the PAH gene were directly sequenced with Sanger method. RESULTS: PAH gene mutations were detected in all of the 102 alleles of the patients, which included 31 types of mutations. Common mutations included R243Q (17/102, 16.67%), IVS4-1G to A (9/102, 8.82%), R241C (8/102, 7.84%), R111X (8/102, 7.84%), and V399V (8/102, 7.84%). In addition, two novel mutations, D101N, 345-347del, have been detected. The 31 types of mutations included missense, nonsense, deletion, and splicing mutations, which were mainly located in exons 7 (29, 28.43%), 11 (18, 17.65%), 3 (16, 15.69%) and 12 (13, 12.75%). CONCLUSION: Mutations of the PAH gene in Linyi region mainly distributed in exons 7, 11, and 3, and the most common mutation were R243Q. Two novel mutations, D101N and 345-347del, have been detected.

Functional and structural characterisation of 5 missense mutations of the phenylalanine hydroxylase.

Phenylketonuria (PKU) and hyperphenylalaninemia (HPA) are a group of genetic disorders predominantly caused by mutations in the phenylalanine hydroxylase (PAH) gene. To date, more than 950 variants have been identified, however the pathogenic mechanism of many variants remains unknown. In this study, in silico prediction and in vitro prokaryotic and eukaryotic expression systems were used to functionally characterize five PAH missense variants (p.F233I, p.R270I, p.F331S, p.S350Y, and p.L358F) previously identified in Slovak and Czech patients. p.F233I, p.R270I, and p.S350Y were classified as deleterious mutations since they showed no specific activity in functional assay and no response to chaperone co-expression. Protein levels of these PAH variants were very low when expressed in HepG2 cells, and only p.S350Y responded to BH4 precursor overload by significant increase in PAH monomer, probably due to reduced rate of protein degradation as the result of proper protein folding. Variants p.F331S and p.L358F exerted residual enzymatic activity in vitro. While the first can be classified as probably pathogenic due to its very low protein levels in HepG2 cells, the latter is considered to be mild mutation with protein levels of approximately 17.85% compared to wt PAH. Our findings contribute to better understanding of structure and function of PAH mutated enzymes and optimal treatment of PKU patients carrying these mutations using BH4 supplementation.

Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

BACKGROUND: Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. METHODS: In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). RESULTS: Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. CONCLUSIONS: Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

In vitro residual activity of phenylalanine hydroxylase variants and correlation with metabolic phenotypes in PKU.

Hyperphenylalaninemias (HPAs) are genetic diseases predominantly caused by a wide range of variants in the phenylalanine hydroxylase (PAH) gene. In vitro expression analysis of PAH variants offers the opportunity to elucidate the molecular mechanisms involved in HPAs and to clarify whether a disease-associated variant is genuinely pathogenic, while investigating the severity of a metabolic phenotype, and determining how a variant exerts its deleterious effects on the PAH enzyme. To study the effects of gene variants on PAH activity, we investigated eight variants: c.611A>G (p.Y204C), c.635T>C (p.L212P), c.746T>C (p.L249P), c.745C>T (p.L249F), c.809G>A (p.R270K), c.782G>C (p.R261P), c.587C>A (p.S196Y) and c.1139C>T (p.T380M), associated with different phenotypic groups. Transient expression of mutant full-length cDNAs in COS-7 cells yielded PAH proteins with PAH activity levels between 7% and 51% compared to the wild-type enzyme. With one exception (p.Y204C, which had no significant impact on PAH function), lower PAH activity was associated with a more severe phenotype (e.g. p.L249P with 7% PAH activity, 100% of classic PKU and no BH4 responsiveness), while higher activity correlated with milder phenotypes (e.g. p.T380M with 28% PAH activity, 97% of mild HPA and 83% of BH4 responsiveness). The results of the in vitro residual PAH activity have major implications, both for our understanding of genotype-phenotype correlations, and thereby existing inconsistencies, but also for the elucidation of the molecular basis of tetrahydrobiopterin (BH4) responsiveness.

Biochemical analysis of Centaurea depressa phenylalanine ammonia lyase (PAL) for biotechnological applications in phenylketonuria (PKU).

CONTEXT: Phenylketonuria (PKU) is the most common hereditary defect of phenylalanine hydroxylase (PAH) enzyme achieving the hydroxylation of phenylalanine (Phe). Phenylalanine ammonia lyase (PAL) converts Phe to a harmless metabolite, trans-cinnamic acid (TCA) in plants and PAL enzyme activity is fairly high in plants rich in flavonoids. OBJECTIVE: The study aimed the biochemical analysis of PAL form Centaurea depressa BIEB. (Asteraceae) a flavonoid rich plant. This study may form the main frame of future research efforts for the development of a plant preparation aimed for oral intake in PKU patients in an attempt to enrich their diet by allowing them to ingest some food stuff containing Phe without being exposed to complications. MATERIALS AND METHODS: PAL was partially purified from the leaves of C. depressa. Enzyme activity was determined in comparison with that of other herbs that reportedly have a high PAL activity. Enzyme optimization was achieved and the PAL protein was detected by western blotting. RESULTS: C. depressa PAL demonstrated high activity (34.9 ± 0.6 U/mg protein). The enzyme was purified by 1.92-fold, which resulted in an activity of 53.30 ± 0.2 U/mg protein. The high-performance liquid chromatography analyzes of the PAL activity both before and after purification were in agreement. Western blot of PAL exhibited a 70 kDa protein band. The optimum pH and temperature are pH 8.8 and 37 °C. The optimum activities under gastric and intestinal digestion conditions were observed at pH 4.0 and pH 8.0, respectively. DISCUSSION AND CONCLUSION: PAL activity of C. depressa is high, and does not disappear under different environmental conditions. This enzyme could be used for the development of dietary foods and biotechnological products for patients with PKU.

Genetic background of hyperphenylalaninemia in Nagasaki, Japan.

Phenylketonuria (PKU) and related hyperphenylalaninemia (HPA) are caused by a deficiency in hepatic phenylalanine hydroxylase (PAH). The incidence of PKU in Nagasaki prefecture is higher than that in all parts of Japan (1/15 894 vs 1/120 000). To investigate the genetic background of patients with HPA in Nagasaki prefecture, mutation analysis was done in 14 patients with PKU or mild HPA. Homozygous or compound heterozygous PAH mutations were identified in all the patients. The spectrum of PAH mutations in the cohort was broad and similar to those in all parts of Japan and East Asian countries. R53H is the most common mutation in patients with mild HPA. The present results provide further support for genotype-phenotype correlations in patients with HPA. The high incidence of PKU in Nagasaki, the westernmost part of Japan, might be due to migration of people with PAH mutations from China and Korea, and geographic factors.

Endothelial cell tetrahydrobiopterin deficiency attenuates LPS-induced vascular dysfunction and hypotension.

Overproduction of nitric oxide (NO) is thought to be a key mediator of the vascular dysfunction and severe hypotension in patients with endotoxaemia and septic shock. The contribution of NO produced directly in the vasculature by endothelial cells to the hypotension seen in these conditions, vs. the broader systemic increase in NO, is unclear. To determine the specific role of endothelium derived NO in lipopolysaccharide (LPS)-induced vascular dysfunction we administered LPS to mice deficient in endothelial cell tetrahydrobiopterin (BH4), the essential co-factor for NO production by NOS enzymes. Mice deficient in endothelial BH4 production, through loss of the essential biosynthesis enzyme Gch1 (Gch1(fl/fl)Tie2cre mice) received a 24hour challenge with LPS or saline control. In vivo LPS treatment increased vascular GTP cyclohydrolase and BH4 levels in aortas, lungs and hearts, but this increase was significantly attenuated in Gch1(fl/fl)Tie2cre mice, which were also partially protected from the LPS-induced hypotension. In isometric tension studies, in vivo LPS treatment reduced the vasoconstriction response and impaired endothelium-dependent and independent vasodilatations in mesenteric arteries from wild-type mice, but not in Gch1(fl/fl)Tie2cre mesenteric arteries. Ex vivo LPS treatment decreased vasoconstriction response to phenylephrine in aortic rings from wild-type and not in Gch1(fl/fl)Tie2cre mice, even in the context of significant eNOS and iNOS upregulation. These data provide direct evidence that endothelial cell NO has a significant contribution to LPS-induced vascular dysfunction and hypotension and may provide a novel therapeutic target for the treatment of systemic inflammation and patients with septic shock.