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• Volume and hematocrit of blood applied to filter paper collection devices affects test results. • Volume of blood assumed to be in 3.2 mm filter paper sub-punch is incorrect resulting in bias. • Volumetric devices negate these pre-analytical issues, improving precision and accuracy. • Devices integrate easily into the workflow of the routine screening/metabolic laboratory. • Volumetric devices will improve biochemical monitoring of patients with Phenylketonuria. Measurement of dried blood spot (DBS) phenylalanine (Phe) is central to the monitoring of patients with phenylketonuria. However, the volume and hematocrit (Hct) of the blood applied to conventional DBS cards significantly affects analytical results. Volumetric blood collection devices are reported to be more accurate, precise and less prone to Hct effects. Accuracy, imprecision, effect of blood volume and Hct were evaluated for measurement of Phe and tyrosine using three volumetric devices and compared with the conventional PerkinElmer-226 filter-paper collection devices. i.e. conventional DBS cards. Applicability for use in a clinical laboratory was assessed qualitatively. Blood volume did not impact on the performance of the volumetric devices; however, significant biases were observed with the conventional DBS card. A higher Hct introduced unacceptable bias for Neoteryx-Mitra and conventional DBS card. All devices had a mean relative standard deviation (RSD) ≤ 4.1 %, except for the Neoteryx-Mitra (≤ 6.2 %). Relative to liquid blood, the mean biases of Phe for the various devices were −5.1 (HemaXis-DB10), −7.8 (Capitainer-qDBS), −12.0 (Neoteryx-Mitra) and −32.6 % (conventional DBS card). Introducing volumetric collection devices will overcome the significant pre-analytical issues associated with conventional DBS collection and improve the biochemical monitoring of patients with PKU. [ABSTRACT FROM AUTHOR]

We employed a Sendai virus-based reprogramming method to transform human lymphoblastoid cell lines (LCL) derived from two individuals diagnosed with phenylketonuria (PKU) into induced pluripotent stem cells (iPSC). This reprogramming process involved the expression of the four Yamanaka factors: KLF4, OCT4, SOX2, and C-MYC. The resulting patient-specific iPSCs exhibited a normal karyotype and expressed endogenous pluripotent markers NANOG and OCT-4. Notably, these iPSCs demonstrated strong differentiation capabilities, giving rise to cell populations representing the ectoderm, endoderm, and mesoderm germ layers. [ABSTRACT FROM AUTHOR]

Phenylketonuria is a rare autosomal recessive metabolic disorder mainly due to a significant reduction in the enzyme phenylalanine hydroxylase, resulting in elevation of phenylalanine in the blood. Here, we have established two fibroblast-derived induced pluripotent stem cell lines using Sendai virus-based reprogramming. The established induced pluripotent stem cell lines exhibited a normal karyotype and expressed markers of pluripotency assessed through quantitative PCR, flow cytometry and immunocytochemistry. These cell lines also demonstrated the ability to differentiate into the three primary germ layers of the human body, including ectoderm, endoderm, and mesoderm. [ABSTRACT FROM AUTHOR]

Background There remains a limited understanding of the metabolic perturbations, beyond phenylalanine (Phe) metabolism, that contribute to phenotypic variability in phenylketonuria (PKU). Objectives This study aimed to characterize changes in the PKU plasma metabolome following a 5-d metabolic camp intervention and to compare PKU profiles with those of matched healthy controls. Methods In 28 females (aged 12–57 y), fasting plasma samples were collected on the first (day 1) and final (day 5) days of camp to measure metabolic control and to complete untargeted metabolomic profiling. Three-day dietary records were collected to assess changes in dietary adherence and composition. Univariate (Wilcoxon signed-rank and Mann–Whitney U test) and multivariate (random forest, hierarchical clustering) analyses were performed to identify clinical and metabolic features that were associated with the intervention and disease state. Results Relative to healthy controls, Phe catabolites, ketones, and carnitine- and glycine-conjugated fatty acids were elevated in females with PKU at baseline, whereas fatty acylcholine metabolites were substantially lower. After the camp intervention, plasma Phe concentrations decreased [median change: –173 µmol/L (IQR: –325, –28 µmol/L)] and 70% of PKU participants demonstrated improved dietary adherence by decreasing Phe intake and/or increasing medical food consumption. This was accompanied by a shift in abundance for 223 metabolites (q  < 0.05). Compounds associated with the metabolism of Phe, fatty acids, and choline contributed most to profile differences between camp days 1 and 5. Conclusions In females with PKU, untargeted metabolomics identified prominent perturbations in amino acid and lipid metabolites associated with bioenergetic impairment and oxidative stress. Choline-conjugated lipids could have fundamental roles in these pathways and they have not been previously evaluated in PKU. A short-term camp intervention was effective for improving or fully normalizing the abundance of the identified discriminatory metabolites. [ABSTRACT FROM AUTHOR]

Abstract Phenylketonuria remains one of the most common inborn errors of metabolism. In the UK it is detected on the newborn heel-prick screening sample allowing early treatment with a strict low phenylalanine diet supplemented with artificial amino acids and appropriate vitamin and minerals. Although the long-term prognosis is good, there is an increasing body of evidence highlighting subtle problems in neuropsychological function with slower reaction times and poorer executive function than peers. White matter changes clearly seen on brain magnetic resonance imaging may have some relationship to these neuropsychological difficulties but their significance is not clearly understood. The diet, although successful, is difficult to follow lifelong and with its attendant risks of nutritional deficiencies needs careful specialist management. In view of these challenges new treatments such as sapropterin (a tetrahydrobiopterin analogue) and large neutral amino acids are currently being used in phenylketonuria and clinical trials are underway using ammonia lyase as enzyme replacement therapy. Maternal phenylketonuria syndrome remains a risk for those who conceive whilst blood phenylalanine is elevated and females must be counselled early in childhood to avoid this risk. [ABSTRACT FROM AUTHOR]

Our study estimated size of impairment for different cognitive functions in early-treated adults with PKU (AwPKU) by combining literature results in a meta-analytic way. We analysed a large set of functions (N = 19), each probed by different measures (average = 12). Data were extracted from 26 PKU groups and matched controls, with 757 AwPKU contributing 220 measures. Effect sizes (ESs) were computed using Glass' ∆ where differences in performance between clinical/PKU and control groups are standardized using the mean and standard deviation of the control groups. Significance was assessed using measures nested within independent PKU groups as a random factor. The weighted Glass' ∆ was − 0.44 for all functions taken together, and − 0.60 for IQ, both highly significant. Separate, significant impairments were found for most functions, but with great variability (ESs from −1.02 to −0.18). The most severe impairments were in reasoning, visual-spatial attention speed, sustained attention, visuo-motor control, and flexibility. Effect sizes were larger with speed than accuracy measures, and with visuo-spatial than verbal stimuli. Results show a specific PKU profile that needs consideration when monitoring the disease. • This is the largest meta-analysis of cognitive deficits in adults with PKU to date. • Significant impairments compared to control are shown for most functions. • The profile of deficits is disease-specific. • Some functions are severely impaired, others are spared or weakly impaired. • The level of impairment (overall EF = −0.44) calls for treatment improvements. [ABSTRACT FROM AUTHOR]

Genetic disorders have been a major concern for public health in China, especially in the rural regions. However, there is little information available about prevalence of many common single-gene disorders in Guizhou Province in the south western part of China. In the present study, we performed a molecular newborn screening for four genetic disorders, including beta-thalassemia (β-thal), glucose-6-phosphate dehydrogenase (G6PD) deficiency, phenylketonuria (PKU), and non-syndromic hearing loss and deafness (NSHL) in this region. A total of 515 newborns were genotyped using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) developed for screening the mutations causing these four disorders, and then confirmed by Sanger sequencing. The results showed that 48 out of 515 newborns were carriers of mutations related to these four diseases, with a frequency of 1 in 11 (9.32%). The carrier frequencies for each disease are: β-thal 2.72%; G6PD deficiency 1.94%; PKU 0.78% and NSHL 4.47%. The genotyping results by MALDI-TOF MS were concordant with Sanger sequencing results within 30 randomly selected samples. This is the first study that reveals carrier frequencies of these four diseases in Guizhou Province. These data provide valuable information for the genetic counseling and disease prevention in Guizhou and southwest China. [ABSTRACT FROM AUTHOR]

Phenylketonuria remains one of the most common inborn errors in the United Kingdom. It is detected on the newborn heel-prick screening sample allowing early treatment with a strict low phenylalanine diet supplemented with artificial amino acids, and appropriate vitamin and minerals. Although the long-term prognosis is good, there is an increasing body of evidence highlighting subtle problems in neuropsychological function with slower reaction times and poorer executive function than peers. White matter changes clearly seen on brain magnetic resonance imaging may have some relationship to these neuropsychological difficulties but their significance is not clearly understood. The diet, although successful, is difficult to follow lifelong and with its attendant risks of nutritional deficiencies needs careful specialist management. In view of these challenges new treatments such as sapropterin (a tetrahydrobiopterin analogue) and large neutral amino acids are currently being used in phenylketonuria and a human trial has started using ammonia lyase as enzyme replacement therapy. Maternal phenylketonuria syndrome remains a risk for those who conceive whilst blood phenylalanine is elevated and females must be counselled early in childhood to avoid this risk. [ABSTRACT FROM AUTHOR]

Objectives Regular and accurate monitoring of blood phenylalanine (Phe) and tyrosine (Tyr) levels is prerequisite for a successful management of patients with hyperphenylalaninemia (HPA). We aimed to compare the tandem mass spectrometry (MS/MS) and the amino acid analyzer (AAA) as methods to measure blood Phe and Tyr levels and Phe/Tyr ratio. Methods Venous blood samples were collected for the AAA analysis, using Pinnacle PCX (Pickering Laboratories), with HPLC Series 1200 (Agilent). Capillary blood was spotted directly on filter paper (Whatman 903) for the MS/MS analysis, using 3200 QTrap AB SCIEX and Perkin Elmer Series 200 HPLC system. The Bland–Altman test was used to compare agreement between the methods and Pearson correlation coefficient to assess the association between the methods. Results 207 pairs of measurements were performed. The Phe levels (range 0–2500 μM) obtained by the MS/MS were on average 26.1% (SD 13.9%) lower compared to those obtained by the AAA. The Tyr levels by the MS/MS were on average 15.5% (SD 20.6%) lower. The Phe/Tyr ratio by the MS/MS was on average 10.6% (SD 15.9%) lower. The Pearson correlation coefficients for Phe (range 0–2500 μM), Tyr and the Phe/Tyr ratio were 0.984 ( p < 0.001), 0.841 ( p < 0.001) and 0.987 ( p < 0.001) respectively. Conclusions When monitoring blood Phe and Tyr levels in patients with HPA, clinicians need to be informed about the method used. Due to the considerable inter-assay variability, a single method is preferable for long-term follow-up of patients. When using MS/MS, on average 26% lower blood Phe levels were obtained as compared to the AAA. The guidelines and recommendations on HPA management should take into consideration the differences in laboratory methods. [ABSTRACT FROM AUTHOR]

Phenylketonuria (PKU) is an inborn error of metabolism that impairs the function of the enzyme phenylalanine hydroxylase. Historical treatment includes limiting dietary phenylalanine (Phe) consumption while supplementing with medical food; however, this treatment has been associated with complications, such as nutritional deficiencies and disruptions in the gut microbiota. The study aim was to compare dietary and gut microbiome differences between adult patients on a traditional PKU diet with those receiving the enzyme substitution therapy Palynziq on a liberalized diet while controlling blood Phe levels to <600 μmol/L (to convert to mg/dL divide by 60.5). A cross-sectional study was conducted comparing patients on a traditional Phe-restricted diet with patients receiving Palynziq eating a liberalized diet. Six patients eating a traditional Phe-restricted diet with medical food and 6 patients on Palynziq eating a liberalized diet without medical food intake for more than 3 years were selected from the University of Kentucky Metabolic Clinic from August to December 2019. Nutrient intake from 3-day diet records and fecal microbiome taxonomic abundances were analyzed. Mann-Whitney U tests were used for dietary data analysis. Differential abundance analysis for microbiome taxa and pathway data was done using DESeq2 analysis. Dietary data showed patients receiving Palynziq consumed a lower percent of kilocalories from total protein and lower amounts of most micronutrients, but consumed greater amounts of intact protein and cholesterol (P <.05). Microbiome data revealed a greater abundance of the phylum Verrucomicrobia and genus Lachnobacterium in the Traditional group and a greater abundance of the genus Prevotella in the Palynziq group (P <.05). Pathway analysis depicted greater enrichment in carotenoid and amino acid metabolism pathways in the Traditional group (P <.05). Protein (% kcal), dietary fiber (g), fat (% kcal), linolenic acid (% Dietary Reference Intakes), and age were correlated with the underlying microbial community structure for both groups combined. Patients with PKU treated with Palynziq on a liberalized diet manifest significant differences in diet composition compared with those treated with traditional Phe-restricted diets. Several of these dietary differences may affect the microbiome architecture. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Objectives: Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism due to a defect in phenylalanine hydroxylase (PAH). Treatment principle is to reduce phenylalanine concentration sufficiently to prevent neuropathological effects. Dietary management is performed and the effect of treatment is monitored by regular analysis of phenylalanine and tyrosine. The aim of the study was to develop a rapid method to routinely measure both metabolites in minimal bloodspot volume (1.5mm Ø, corresponding with a volume of 1.3μL blood). Method: Whole blood was spiked with phenylalanine and tyrosine at 24 different concentrations. Dried blood spots (DBS) were prepared, after which punches of 1.5mm Ø and 6mm Ø (corresponding with a volume of 12.4μL) were taken. Additionally, punches of both sizes were prepared from DBS of PKU-patients (n=77). All samples were analyzed by tandem mass-spectrometry and results between both punches were compared. Results: A good correlation between concentrations of phenylalanine and tyrosine in 1.5 and 6mm punches was found (r2 =0.9917 and r2 =0.9892, respectively). Analysis of phenylalanine and tyrosine in punches of PKU-patients (n=77) showed similar results and fitted within the procentual range of the between run variation. Conclusion: We developed an accurate and rapid method to analyze phenylalanine and tyrosine concentrations in a 1.5mm Ø bloodspot punch with an estimated whole blood volume of 1.3μL. This technical improvement does not only result in a 10 fold reduction in required patients' material, but also in a 30–60min time saving in sample preparation. [ABSTRACT FROM AUTHOR]

Abstract: Tetrahydrobiopterin (BH4) deficiency is a rare, congenital and lethal condition resulting in phenylalanine build-up that can lead to mental retardation and developmental defects, unless properly treated. About 1 million newborn infants in Japan undergo neonatal PKU screening every year, of which about 1 in 2 million are diagnosed with the condition. In this post-marketing surveillance study, 19 patients with BH4 deficiency in whom BH4 supplementation with sapropterin dihydrochloride (Biopten®) (hereafter referred to as ‘BH4 therapy’) was initiated before the age of 4years, were followed up for ⩽28years. Patients who screened positive for BH4 deficiency were treated with supplemental BH4 plus L-dopa and 5-hydroxytryptophan. Data on the patients’ clinical courses were collected once yearly at 10 medical centers in Japan. Seventeen patients were diagnosed with 6-pyruvoyl tetrahydropterin synthase deficiency and two with dihydropteridine reductase deficiency at an average age of 3.6months; the mean age at end of follow-up was 14.6years. Average duration of BH4 therapy (mean dose, 5mg/kg per day) was 13.2years. Serum phenylalanine was reduced from more than 10mg/dL at the start of drug administration to less than 2mg/dL at end of follow-up. No abnormalities in height or weight were observed in any patients, except for one female patient with familial obesity. No unwarranted side effects were reported throughout the long-term course of treatment, even during pregnancy. BH4 therapy can effectively maintain serum phenylalanine levels within the normal range in patients with BH4 deficiency, and demonstrated excellent long-term safety, with no side effects. [Copyright &y& Elsevier]

Abstract: Phenylketonuria is an autosomal recessive inborn error of metabolism resulting from phenylalanine hydroxylase deficiency. Genetic basis of phenylalanine hydroxylase deficiency has been reported in various European and Asian countries with few reports available in Arab populations of the Mediterranean region. This is the first pilot study describing phenotype and genotype of 23 Lebanese patients with phenylketonuria. 48% of the patients presented mainly with neurological signs at a mean age of 2years 9months, as newborn screening is not yet a nationwide policy. 56.5% of the patients had classical phenylketonuria. Thirteen different mutations were identified: splice site 52%, frameshift 31%, and missense 17% with no nonsense mutations. IVS10-11G>A was found mainly in Christians at high relative frequency whereas Muslims carried the G352fs and R261Q mutations. A rare splice mutation IVS7+1G>T, not described before, was identified in the homozygous state in one family with moderate phenylketonuria phenotype. Genotype–phenotype correlation using Guldberg arbitrary value method showed high consistency between predicted and observed phenotypes. Calculated homozygosity rate was 0.07 indicating the genetic heterogeneity in our patients. Our findings underline the admixture of different ethnicities and religions in Lebanon that might help tracing back the PAH gene flux history across the Mediterranean region. [Copyright &y& Elsevier]

Abstract: Background: Previous research has suggested an increased risk for individuals with phenylketonuria (PKU) of developing depression and other mood disorders. As PKU can disrupt neurotransmitter synthesis via biochemical mechanisms, depressive symptoms are hypothesised to result from neurotransmitter dysregulation. Whilst adherence (or return) to the phenylalanine-restricted diet may resolve or improve symptoms of depression, data to demonstrate a direct relationship between biochemistry and mood in this population are lacking. Methods: Thirteen adolescents with early and continuously treated PKU and eight sibling controls were compared in their total reported depressive symptoms. A general executive function assessment was also undertaken in the PKU group. Correlations between depressive symptoms and biochemical markers were examined within the PKU group only. Results: Correlational analyses within the PKU group demonstrated strong and significant associations between depressive symptoms and long term exposure to either a high phenylalanine:tyrosine ratio, or low tyrosine. Increasing symptoms of depression were also found to be associated with poorer executive function in the PKU sample. However, both groups of adolescents scored within the normal range in symptoms of depression (p >0.05). Conclusions: Significant associations were observed between biochemical markers indicating poorer dietary control and increasing depressive symptoms in a sample of adolescents with early and continuously treated PKU, although symptoms of depression remained within the normal range. An association between depressive symptoms and poorer EF was also demonstrated. Further research is needed to establish whether the depressive symptoms observed in this young population represent an emerging (subclinical) risk for major depressive disorder as they age. [Copyright &y& Elsevier]

Abstract: Phenylketonuria remains one of the most common inborn errors in the United Kingdom. It is detected on the newborn heel-prick screening sample allowing early treatment with a strict low phenylalanine diet supplemented with artificial amino acids, and appropriate vitamin and minerals. Although the long-term prognosis is good, there is an increasing body of evidence highlighting subtle problems in neuropsychological function with slower reaction times and poorer executive function than peers. White matter changes clearly seen on brain magnetic resonance imaging may have some relationship to these neuropsychological difficulties but their significance is not clearly understood. The diet, although successful, is difficult to follow lifelong and with its attendant risks of nutritional deficiencies needs careful specialist management. In view of these challenges new treatments such as sapropterin (a tetrahydrobiopterin analogue) and large neutral amino acids are currently being used in phenylketonuria and a human trial has started using ammonia lyase as enzyme replacement therapy. Maternal phenylketonuria syndrome remains a risk for those who conceive whilst blood phe is elevated and females must be counselled early in childhood to avoid this risk. [Copyright &y& Elsevier]

Abstract: Objective: : The knowledge of the molecular basis of the Phenylketonuria (PKU, MIM# 261600) in different countries provides relevant information for undertaking specific and rational mutation detection strategies in each population and for the implementation of adequate dietary and cofactor treatment. There are no data available in Moroccan population. Design and methods: : In this work we describe the genetic analysis by mutation scanning using denaturing gradient gel electrophoresis (DGGE) and subsequent direct sequencing of 20 different PKU families from Morocco. We have also included the study of 7 Moroccan PKU patients living in Spain detected by the Spanish newborn screening program. Results: : The mutational spectrum in the first sample included eight different changes, one of them, p.K85X, was novel. The most common mutation was the frame shift change p.G352fsdelG identified in 62.5% of the mutant chromosomes studied. Other changes (p.R176X, IVS10nt-11 g>a, p.W120X, p.A165T, p.R243X and p.R243Q) were identified, respectively, in 2 or 3 mutant alleles. All detected mutations were severe according to the classical phenotype of the patients. In the 7 patients living in Spain we have detected 4 severe mutations (p.G352fs, p.R176X, Y198fs and Exon3del) and also milder changes such as p.A403V, p.S196T, p.D145V and p.R408Q detected in 3 mild hyperphenylalaninemia (MHP) patients and a novel p.L258P found in a mild PKU patient. Conclusion: : The results provide important information on the distribution of PKU mutations in this Mediterranean area gaining insight into the genetic epidemiology of the disease. [Copyright &y& Elsevier]

Abstract: Hyperphenylalaninemia is a group of autosomal recessive disorders caused by a wide range of phenylalanine hydroxylase (PAH) gene variants. To study the effects of mutations on PAH activity, we have reproduced five mutations (p.N223Y, p.R297L, p.F382L, p.K398N and p.Q419R) that we recently identified in a population of Southern Italy. Transient expression of mutant full-length cDNAs in human HEK293 cells yielded PAH variants whose l-phenylalanine hydroxylase activity was between 40% and 70% that of the wild-type enzyme. Moreover, Western blot analysis revealed a 50-kD monomer in all mutants thereby indicating normal synthesis of the mutant proteins. Because of the clinical mild nature of the phenotypes we performed an in vivo BH4 loading test. This was positive in all tested patients, which indicates that they are likely to respond to the coenzyme in vivo. We also analysed the environment of each mutation site in the available crystal structures of PAH by using molecular graphics tools. The structural alteration produced by each mutation was elucidated and correlated to the mutated properties of the mutant enzymes. All the data obtained demonstrate the disease-causing nature of the five novel variants. [Copyright &y& Elsevier]

Abstract: Phenylketonuria (PKU) is a disease characterized by an inability to metabolize the amino acid l-phenylalanine. The resulting buildup leads to brain damage and ultimately mental retardation in children if their phenylalanine intake is not carefully controlled. The National Institutes of Health recently suggested that people with PKU monitor their phenylalanine levels throughout their life and be put on a low phenylalanine diet. As an alternative approach to analysis using blood, this paper describes the first reagentless dehydrogenase based sensor for the determination of phenylalanine in human urine. The clinical range of phenylalanine in human urine is 20–60mM for people with PKU. Although most clinical analysis is performed using blood, urine was chosen due to its high concentrations of phenylalanine in phenylketonurics, as well as its simple, safe, and painless collection. The sensor is comprised of a carbon paste electrode with nicotinamide adenine dinucleotide (NAD+), phenylalanine dehydrogenase (PDH), uricase, and an electron mediator, 3,4-dihydroxybenzaldehyde (3,4-DHB), all mixed into the paste. The electron mediator reacts with the electrode surface to produce two redox species, which catalytically oxidize NADH. The behavior of the electron mediator mixed into a carbon paste electrode has not been previously investigated. Cyclic voltammetry was used to characterize the sensor''s response to NADH, and with the addition of PDH and NAD+ to the paste, its response to phenylalanine in human urine. The limit of detection for phenylalanine is 0.5mM (S/N=3). [Copyright &y& Elsevier]

Abstract: Background: Multiplex ligation probe amplification (MLPA) is a sensitive and efficient technique for molecular diagnosis of diseases involving deletions or duplications of large genomic regions. In phenylketonuria (PKU), most of the mutant alleles correspond to missense mutations and large deletions have been scarcely identified. In this study, we report for the first time the use of MLPA analysis on PKU patients to detect exonic deletions. Method: DNA from 22 unrelated PKU patients with an incomplete genetic diagnosis after standard mutation detection analysis were subjected to MLPA analysis. Deletions were confirmed by long-range PCR and sequence analysis. Results: The technique identified two large genomic deletions in the phenylalanine hydroxylase (PAH) gene, of 6.6 kb and 1.8 kb, including exons 3 and 5, respectively. The chromosomal breakpoints were established by long-range PCR and chromosomal walking, confirming the involvement of repetitive sequences in the deletions. Conclusion: MLPA may complement routine mutation screening in PKU patients, although, in the sample studied, exonic deletions in the PAH gene do not appear to be a frequent cause of PKU. [Copyright &y& Elsevier]

Abstract: Objective: The aim of the present study was to examine the nature of previously reported deficits in sustained attention and response inhibition in adults with the developmental disorder, phenylketonuria (PKU). Methods: This study used event-related potentials (ERPs) to examine the performance of PKU adults (n =9) and a matched control group (n =9) on a visual Go-Nogo task. Results: Comparison of behavioural measures between the PKU and control groups failed to reach statistical significance, yet analysis of the ERPs showed statistically significant amplitude reductions in the P1 and N1 components elicited following presentation of stimuli, and a reduction in the amplitude of the N2 component elicited following presentation of Nogo stimuli. Conclusions: These results suggest that adults with PKU, despite being continuously treated from birth, manifest subtle impairments in distinct aspects of information processing including early sensory processing of visually presented information, as well as impairments in inhibitory functions. Significance: The results contribute to an understanding of the neurophysiological mechanisms that are implicated in PKU and highlight the sensitivity of ERP techniques for the identification of the loci of information processing deficits in clinical groups. [Copyright &y& Elsevier]

Abstract: Background: Phenylketonuria (PKU) is an inborn error of metabolism that results from a deficiency of phenylalanine hydroxylase (PAH). We demonstrated PAH mutational spectrum from patients with PKU, including 10 novel and 3 tetrahydrobiopterin (BH4)-responsive mutations. In this study, 11 PAH missense mutations, including 6 novel mutations (P69S, G103S, L293M, G332V, S391I, A447P) found in our previous study, 2 mutations common in east Asian patients with PKU (R243Q, R413P), and 3 tetrahydrobiopterin (BH4)-responsive mutations (R53H, R241C, R408Q) have been functionally and structurally analyzed. Methods: A transient protein overexpression system and an in vitro BH4-responsiveness study were used. The effects of PAH missense mutations on the PAH protein structure were also analyzed. To determine the conservation of 12 mutated residues, PAH was aligned using BLAST against full genomic sequences of 221 different species. Model structures of PAH protein and the composite tetramer were constructed using the software program, SHEBA. Results: No PAH activity was detected for some mutants. However, the residual activities associated with other mutants ranged over a wide spectrum. The missense mutations responsive to BH4 were not highly conserved throughout the 43 species in the multiple sequence alignment that encode PAH. The composite model structure of PAH revealed that dimer stability was reduced in the BH4-responsive mutants, whereas tetramer stability remained normal. Conclusion: This expression study analyzed PAH mutations and model structures of mutant PAH proteins are proposed. Correlation between the proposed mutant PAH structures and functions are suggested. [Copyright &y& Elsevier]

Abstract: The large neutral amino acid transporter type 1, LAT1, is the principal neutral amino acid transporter expressed at the blood–brain barrier (BBB). Owing to the high affinity (low K m) of the LAT1 isoform, BBB amino acid transport in vivo is very sensitive to transport competition effects induced by hyperaminoacidemias, such as phenylketonuria. The low K m of LAT1 is a function of specific amino acid residues, and the transporter is comprised of 12 phylogenetically conserved cysteine (Cys) residues. LAT1 is highly sensitive to inhibition by inorganic mercury, but the specific cysteine residue(s) of LAT1 that account for the mercury sensitivity is not known. LAT1 forms a heterodimer with the 4F2hc heavy chain, which are joined by a disulfide bond between Cys160 of LAT1 and Cys110 of 4F2hc. The present studies use site-directed mutagenesis to convert each of the 12 cysteines of LAT1 and each of the 2 cysteines of 4F2hc into serine residues. Mutation of the cysteine residues of the 4F2hc heavy chain of the hetero-dimeric transporter did not affect transporter activity. The wild type LAT1 was inhibited by HgCl2 with a K i of 0.56±0.11 μM. The inhibitory effect of HgCl2 for all 12 LAT1 Cys mutants was examined. However, except for the C439S mutant, the inhibition by HgCl2 for 11 of the 12 Cys mutants was comparable to the wild type transporter. Mutation of only 2 of the 12 cysteine residues of the LAT1 light chain, Cys88 and Cys439, altered amino acid transport. The V max was decreased 50% for the C88S mutant. A kinetic analysis of the C439S mutant could not be performed because transporter activity was not significantly above background. Confocal microscopy showed the C439S LAT1 mutant was not effectively transferred to the oocyte plasma membrane. These studies show that the Cys439 residue of LAT1 plays a significant role in either folding or insertion of the transporter protein in the plasma membrane. [Copyright &y& Elsevier]

Abstract: Background:: Phenylketonuria (PKU), an inborn error of metabolism, is treated with a low phenylalanine (Phe) lifelong diet, which can be characterized as vegetarian. 8-Hydroxy-2-deoxyguanosine (8-OHdG) is highly implicated in degenerative diseases. Objective:: To evaluate the effect of plasma total antioxidant status (TAS) and Phe on the serum marker of DNA damage, 8-OHdG, in PKU. Methods:: Twenty-four PKU patients on a strict diet (group A), 25 PKU patients on a “loose diet” (group B), and 24 healthy children (controls) participated in this study. Plasma TAS was evaluated spectrophotometrically. 8-OHdG and Phe were measured in blood with immunoassays. Results:: TAS levels were significantly higher (P < 0.001) in group A (1458 ± 140 μmol/L) and controls (1452 ± 235 μmol/L) than those in group B (907 ± 150 μmol/L). In contrast, 8-OHdG serum levels were 2-fold higher in group B (0.22 ± 0.03 ng/mL) as compared with those in group A (0.11 ± 0.02 ng/mL) and 3-fold higher than those in controls (0.08 ± 0.02 ng/mL) (P < 0.001). As expected, Phe levels were also significantly higher in group B than those in the other study groups. Positive correlation coefficients were found between Phe and 8-OHdG levels, whereas negative correlations were evaluated between TAS and 8-OHdG in all groups. Conclusions:: The high Phe and the low TAS plasma levels in PKU patients on a “loose diet” may induce DNA oxidation, as evidenced by the measured high 8-OHdG level in their sera. 8-OHdG evaluation may be a useful marker of increased risk for a neurodegenerative process. [Copyright &y& Elsevier]

Abstract: Objectives:: To investigate the BH4 response in a group of patients with phenylketonuria (PKU) in order to offer this alternative treatment to the responsive patients. Design and methods:: The 24-h-long Phe/BH4 loading test was performed on 64 PKU patients requiring dietary treatment. Results:: All patients with mild-PKU and 75% of patients with moderate-PKU were BH4 responsive, while only 11% of classic-PKU patients showed good/partial response (P < 0.0001). The percentages of Phe decrease after the BH4 loading test were significantly different in the three PKU phenotypes (mild PKU: 67.9 ± 18.7; moderate PKU: 37.4 ± 16.8; and classical PKU: 21.9 ± 13.7; ANOVA with Bonferroni correction: P < 0.0001). We report four mutations (P147S, D222G, P275S, and P362T) not previously associated with BH4 responsiveness, all of them combined with mutations with zero predicted residual activity. Conclusion:: Both the percentage of Phe decrease and the Phe value achieved 24 h after BH4 loading are valuable data in predicting a response. We report four mutations not previously associated with BH4 responsiveness. [Copyright &y& Elsevier]

Background: Serum neural protein S-100B concentration is considered as a marker of CNS lesions. Phenylketonuria (PKU) is a metabolic disorder characterized by high phenylalanine (Phe) levels in blood and foci of myelin absence in the CNS of untreated patients.Aim: To evaluate S-100B blood levels in PKU patients.Methods: Twenty-five (N = 25) PKU patients of comparable age, who were diagnosed by neonatal screening and “followed up” in our Inborn Error of Metabolism Department, were divided into two groups: group A (N = 13) with almost normal Phe levels and group B (N = 12) “off diet” with high Phe concentrations. Their MRI examinations were normal 12–14 months before the beginning of the study. Twenty-three (N = 23) healthy children were the controls. Serum S-100B levels, measured with an immunoluminometric assay, were greatly elevated in the group B (0.48 ± 0.6 μg/l) as compared to those of group A (0.16 ± 0.4 μg/l, P < 0.001) and controls (0.10 ± 0.02, P < 0.001). Positive correlation was found between S-100B and Phe blood concentration (r = 0.46, P < 0.01). Foci of myelin absence in MRIs were observed in 1/13 of group A and in 10/12 of group B at the end of this study.Conclusions: (a) Serum S-100B protein level, for the first time evaluated in PKU, was positively correlated with Phe blood level in PKU patients. (b) S-100B blood estimation could be a useful peripheral marker of CNS lesions in patients with demyelinated disease such as PKU. [Copyright &y& Elsevier]

Objective: a) To evaluate acetylcholinesterase (AChE) activities in erythrocyte membranes from phenylketonuric (PKU) patients and controls and to correlate with their plasma phenylalanine (Phe), tyrosine (Tyr), alanine (Ala) and dopamine (DA) levels. b) To determine the in vitro effects of Phe, Ala and Phe plus Ala on their AChE activities.Design and Methods: AChE activities were determined spectrophotometrically in erythrocyte membranes from PKU children (n = 12) adhering to their diet (group A), from 11 “off diet” (group B) and from 23 controls. Their plasma amino acids were evaluated with an amino acid analyser and DA with an HPLC method. Ala (1.8 mM) and/or Phe (1.8 mM) were added in the enzyme incubation medium from controls, whereas only Ala was added in that from group B.Results: AChE activity (1.19 ± 0.05 ΔOD/min × mg protein), Tyr (46 ± 17 μmol/L) and DA (56 ± 18 μmol/L) were remarkably decreased by about 60% in group B as compared to those of group A (3.01 ± 0.18 ΔOD/min × mg protein, 115 ± 39 μmol/L, 137 ± 29 μmol/L, respectively, p < 0.001) and controls (3.13 ± 0.16 ΔOD/min × mg protein, 117 ± 44 μmol/L, 142 ± 22 μmol/L, respectively, p < 0.001). Phe negatively correlated with AChE activity and positively with plasma Tyr and DA. Ala reversed the inhibited AChE by Phe in erythrocyte membranes from healthy children to control values, whereas no reverse effect was observed on the enzyme activity from PKU patients.Conclusions: a) The low levels of DA and its precursor Tyr are due to the high Phe blood levels, as a consequence of the decreased activity of Phe-hydroxylase in the liver of our patients. So, high Phe blood levels inhibit AChE in PKU patients, probably resulting in higher acetylcholine concentrations. b) Determination of AChE in erythrocyte membranes from PKU could be a useful marker for the neurotoxic effects of Phe. [Copyright &y& Elsevier]

Background: Women with untreated phenylketonuria (PKU) often have poor reproductive outcomes. Objective: We assessed the effects of intakes of major nutrients on plasma phenylalanine concentrations and we measured phenylalanine hydroxylase activity and phenylalanine intakes in pregnant women with PKU. Design: Dietary intakes and plasma phenylalanine concentrations were compared in 4 subject groups defined on the basis of plasma phenylalanine concentrations: group 1 (n = 23), <360 µmol/L by 10 wk gestation and 120-360 µmol/L throughout the remainder of pregnancy; group 2 (n = 46), <600 µmol/L but not <360 µmol/L by 10 wk gestation and 120-600 µmol/L throughout the remainder of pregnancy; group 3 (n = 24), <600 µmol/L by 10 wk gestation but >600 µmol/L at least once thereafter; group 4 (n = 147), never <600 µmol/L. Results: Except in the first trimester, mean intakes of phenylalanine, energy, and fat tended to be greater in group 1 than in the other groups. The mean protein intake of group 1 tended to be greater than that of the other groups. Intakes of protein (P <0.0001), fat (P <0.0001), and energy (P <0.007) were negatively correlated with maternal plasma phenylalanine concentrations. It appeared that genotype did not affect phenylalanine tolerance. Conclusions: Maternal genotype appeared to have little influence on phenylalanine requirements during the first trimester. Early decline and maintenance of maternal plasma phenylalanine concentrations at <360 µmol/L and mean protein intake greater than the recommended dietary allowance (RDA) with mean energy intake near the RDA resulted in the best reproductive outcomes. Inadequate intakes of protein, fat, and energy may result in elevated plasma phenylalanine concentrations and may contribute to poor reproductive outcomes. [ABSTRACT FROM AUTHOR]

Abstract: Background: Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients. Methods: Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11. Results: A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G>A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11+1G>C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410). Conclusion: With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen. [Copyright &y& Elsevier]

We report a patient with BH4-sensitive phenylketonuria. In neonatal screening, phenylalanine levels above 10 mg/dl were detected. In the tetrahydrobiopterin- (BH4) loading test, phenylalanine concentrations in serum fell significantly. Dihydropteridine reductase activity in blood, pterines, and neurotransmitters in cerebrospinal fluid, as well as pterines in urine were all normal. Mutation analysis revealed compound-heterozygosity for the mutations R408W and K320N. Under BH4-supplementation without a specific phenylalanine-reduced diet, phenylalanine-concentrations are in the therapeutic range and our patient developed normally. [Copyright &y& Elsevier]

Hyperphenylalaninemias (HPAs) are due to autosomal recessive inherited deficiency of phenylalanine hydroxylase and include three different biochemical and clinical phenotypes: classic phenylketonuria, mild phenylketonuria and persistent HPA. Recently the relationship between birth weight and HPA has been investigated. We performed an evaluation of birth weight in our 260 HPA patients. Our results do not support the view that birth weight is reduced in HPA patients and we found no correlation between birth weight and severity of the disease. Only a better knowledge of genetic mechanisms involved in HPA can clarify the interaction between HPA and fetal development. [Copyright &y& Elsevier]

Treatment of phenylketonuria (PKU) consists of restriction of natural protein and provision of a protein substitute that lacks phenylalanine but is enriched in tyrosine. Large and unexplained differences exist, however, in the tyrosine enrichment of the protein substitutes. Furthermore, some investigators advise providing extra free tyrosine in addition to the tyrosine-enriched protein substitute, especially in the treatment of maternal PKU. In this article, we discuss tyrosine concentrations in blood during low-phenylalanine, tyrosine-enriched diets and the implications of these blood tyrosine concentrations for supplementation with tyrosine. We conclude that the present method of tyrosine supplementation during the day is far from optimal because it does not prevent low blood tyrosine concentrations, especially after an overnight fast, and may result in largely increased blood tyrosine concentrations during the rest of the day. Both high tyrosine enrichment of protein substitutes and extra free tyrosine supplementation may not be as safe as considered at present, especially to the fetus of a woman with PKU. The development of dietary compounds that release tyrosine more slowly could be beneficial. We advocate decreasing the tyrosine content of protein substitutes to ≈6% by wt (6 g/100 g protein equivalent) at most and not giving extra free tyrosine without knowing the diurnal variations in the blood tyrosine concentration and having biochemical evidence of a tyrosine deficiency. We further advocate that a better daily distribution of the protein substitute be achieved by improving the palatability of these products. [ABSTRACT FROM AUTHOR]