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Objectives: Patients affected by Phenylketonuria (PKU) require lifelong management based on phenylalanine (Phe) and tyrosine (Tyr) restricted intake or tetrahydrobiopterin (BH4) administration. Frequent monitoring of blood concentration of both amino acids during treatment is the key point for clinicians to achieve the best long-term neuropsychological outcome., Results: The present study develops and validates a rapid and simple method for Phe and Tyr quantization in dried blood spot (DBS) since this specimen has the advantage of being low invasive, easily withdrawn even at home and stable if mail-delivered. The validation studies showed the robustness of the method., Conclusions: Serum and DBS samples from PKU patients were analyzed and compared, finding a good correlation of Phe and Tyr concentrations between the two different matrixes., (© 2013.)

Background: The value of genotyping to predict variant phenotypes in patients with phenylalanine hydroxylase (Pah) deficiency is a matter of debate. However, there exists no comprehensive population relationship study focused on the Han Chinese., Methods: We analyzed genotype-phenotype correlation for 186 different genotypes in 338 unrelated Chinese patients harboring 109 different Pah mutations. Two systems were used in this process. The first was a phenotype prediction system based on arbitrary values (AV) attributed to each mutation. The second was a pair-wise correlation analysis. The observed phenotype for AV analysis was the corresponding metabolic phenotype stratified according to the pretreatment phenylalanine (Phe) value., Results: We found that the observed phenotype matched the predicted phenotype in 54.41% of 272 patients for whom AV information was available; the highest degree of concordance (61.83%) was found in patients with null/null genotypes, whereas the lowest "concordance rate" (32.69%) was observed for patients with expected mild-PKU phenotype. There are repeated inconsistencies for such mutations as R241C, R243Q, R261Q, V388M, V399V, R408Q, A434D and EX6-96A>G which are associated with variable phenotypes in patients with identical genotype. Significant correlations were disclosed between pretreatment Phe values and predicted residual activity (r=-0.45643, P<0.0001) or AV sum (r=-0.59523, P<0.0001)., Conclusion: Our study supports the notion that the Pah mutation genotype is the main determinant of metabolic phenotype in most patients in a particular population, and provided novel insights into the values that underpin the subsequent treatment and the prognosis of PKU in Chinese., (© 2013. Published by Elsevier B.V. All rights reserved.)

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., (© 2013.)

Phenylketonuria (PKU) is a well-defined metabolic disorder arising from a mutation that disrupts phenylalanine metabolism and so produces a variety of neural changes indirectly. Severe cognitive impairment can be prevented by dietary treatment; however, residual symptoms may be reported. These residual symptoms appear to overlap a more prevalent childhood disorder: Attention Deficit/Hyperactivity Disorder (ADHD). However, the aetiology of ADHD is a vast contrast to PKU: it seems to arise from a complex combination of genes; and it has a substantial environmental component. We ask whether these two disorders result from two vastly different genotypes that converge on a specific core phenotype that includes similar dysfunctions of Gray's (Gray, 1982) Behavioural Inhibition System (BIS), coupled with other disorder-specific dysfunctions. If so, we believe comparison of the commonalities will allow greater understanding of the neuropsychology of both disorders. We review in detail the aetiology, treatment, neural pathology, cognitive deficits and electrophysiological abnormalities of PKU; and compare this with selected directly matching aspects of ADHD. The biochemical and neural pathologies of PKU and ADHD are quite distinct in their causes and detail; but they result in the disorder in the brain of large amino acid levels, dopamine and white matter that are very similar and could explain the overlap of symptoms within and between the PKU and ADHD spectra. The common deficits affect visual function, motor function, attention, working memory, planning, and inhibition. For each of PKU and ADHD separately, a subset of deficits has been attributed to a primary dysfunction of behavioural inhibition. In the case of ADHD (excluding the inattentive subtype) this has been proposed to involve a specific failure of the BIS; and we suggest that this is also true of PKU. This accounts for a substantial proportion of the parallels in the superficial symptoms of both disorders and we see this as linked to prefrontal, rather than more general, dysfunction of the BIS., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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 © 2013 Elsevier B.V. All rights reserved.)

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 (1.5 mm Ø, corresponding with a volume of 1.3 μL blood)., Method: Whole blood was spiked with phenylalanine and tyrosine at 24 different concentrations. Dried blood spots (DBS) were prepared, after which punches of 1.5 mm Ø and 6 mm Ø (corresponding with a volume of 12.4 μL) 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 6 mm punches was found (r(2)=0.9917 and r(2)=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 1.5 mm Ø bloodspot punch with an estimated whole blood volume of 1.3 μL. This technical improvement does not only result in a 10 fold reduction in required patients' material, but also in a 30-60 min time saving in sample preparation., (Copyright © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

A trans-National Institutes of Health initiative, Nutrition and Dietary Supplement Interventions for Inborn Errors of Metabolism (NDSI-IEM), was launched in 2010 to identify gaps in knowledge regarding the safety and utility of nutritional interventions for the management of inborn errors of metabolism (IEM) that need to be filled with evidence-based research. IEM include inherited biochemical disorders in which specific enzyme defects interfere with the normal metabolism of exogenous (dietary) or endogenous protein, carbohydrate, or fat. For some of these IEM, effective management depends primarily on nutritional interventions. Further research is needed to demonstrate the impact of nutritional interventions on individual health outcomes and on the psychosocial issues identified by patients and their families. A series of meetings and discussions were convened to explore the current United States' funding and regulatory infrastructure and the challenges to the conduct of research for nutritional interventions for the management of IEM. Although the research and regulatory infrastructure are well-established, a collaborative pathway that includes the professional and advocacy rare disease community and federal regulatory and research agencies will be needed to overcome current barriers., (Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

The mainstream of phenylketonuria (PKU) management is lifelong restriction of protein intake; however, this dietary restriction may be accompanied by insufficient dietary intake of long-chain polyunsaturated fatty acids (LCPUFA). The objective of this review was to assess whether significant depletion of LCPUFA can be detected in PKU patients on low-protein diet and whether LCPUFA supplementation is an effective way to increase the availability of LCPUFA in PKU patients. The method included structured search strategy on Ovid MEDLINE, Scopus, LILACS, and the Cochrane Library CENTRAL databases, with formal inclusion/exclusion criteria, data extraction procedure, and meta-analysis. We evaluated 9 case-control studies and 6 randomized controlled trials, dated from the inception of the databases to 2012. The meta-analysis of the case-control studies showed significantly lower values of both eicosapentaenoic acid and docosahexaenoic acid (DHA) in all biomarkers investigated and that of arachidonic acid in total plasma lipids in PKU patients as compared with healthy controls. There were sufficient data to demonstrate that dietary DHA supplementation of patients with PKU significantly increases the contribution of DHA to total plasma lipids. In summary, suboptimal LCPUFA status, especially that of n-3 LCPUFA, can be detected in PKU patients. Supplementing DHA to the diet of PKU patients may improve their LCPUFA status; however, further research is needed to determine the optimal supplementation dosage and to establish beneficial functional outcomes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Sapropterin dihydrochloride, the synthetic form of 6R-tetrahydrobiopterin (BH4), is an approved drug for the treatment of patients with BH4-responsive phenylketonuria (PKU). The purpose of this study was to assess genotypes and data on the long-term effects of BH4/sapropterin on metabolic control and patient-related outcomes in 6 large European countries., Methods: A questionnaire was developed to assess phenotype, genotype, blood phenylalanine (Phe) levels, Phe tolerance, quality of life, mood changes, and adherence to diet in PKU patients from 16 medical centers., Results: One hundred forty-seven patients, of whom 41.9% had mild hyperphenylalaninemia, 50.7% mild PKU, and 7.4% classic PKU, were followed up over ≤12 years. A total of 85 different genotypes were reported. With the exception of two splice variants, all of the most common mutations were reported to be associated with substantial residual Phe hydroxylase activity. Median Phe tolerance increased 3.9 times with BH4/sapropterin therapy, compared with dietary treatment, and median Phe blood concentrations were within the therapeutic range in all patients. Compared with diet alone, improvement in quality of life was reported in 49.6% of patients, improvement in adherence to diet was reported in 47% of patients, and improvement in adherence to treatment was reported in 63.3% of patients. No severe adverse events were reported., Conclusions: Our data document a long-term beneficial effect of orally administered BH4/sapropterin in responsive PKU patients by improving the metabolic control, increasing daily tolerance for dietary Phe intake, and for some, by improving dietary adherence and quality of life. Patient genotypes help in predicting BH4 responsiveness.

During the last two decades the realization has emerged that the phenotype of the majority of inherited genetic diseases, including inborn errors of metabolism, cannot be predicted by the genotype identified in patients. This is true for PKU and in the majority of fatty acid oxidation (FAO) defects, where the genotypes identified in patients may be allocated into two groups. One comprising big deletions and small out-of-frame deletions/insertions as well as severe splice and stop codon changes, generally giving rise to no or very little protein product, and the other group, comprising small in-frame deletions/insertions and missense variations, resulting in misfolding proteins with varying stability. In all cases of FAO defects the pathophysiology may be due to energy insufficiency as well as toxic effects from accumulated enzyme substrates. In patients carrying missense variations, it may in addition be caused by the presence of misfolding proteins. A common effect of accumulated substrates and misfolding proteins is chronic oxidative stress, the severeness of which may depend on a complex interplay of modifying factors, including genetic, cellular, environmental and dietary. In this review we will discuss the hypothesis that especially the amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS), created in connection with the electron transport chain (ETC), are the driving forces in the balance between cell survival and death. In young and healthy cells small amounts of ROS function as signaling molecules, activating cell protection systems, such as protein quality networks, antioxidant enzymes and metabolic shift from ATP production by the ETC to glycolysis. In the sick and old cell, containing misfolding and damaged proteins, the dynamic range of these protecting systems are narrowed, and cells develop a state of chronic stress, which easier than young and healthy cells may initiate cell death programs like apoptosis and necrosis. We will discuss a wealth of literature that support this hypothesis, which - if supported by studies - is important for new treatment strategies. We conclude that crude antioxidant treatment may not be beneficial, since it may inhibit the survival stress responses. We discuss the ongoing studies to enhance the residual activity of mild misfolding enzyme proteins by cofactor or chemical chaperones or by inducing the transcription of FAO enzyme proteins by bezafibrate with respect to misfolding/distorted conformational proteins ability to create ROS, and the need to know the exact pathophysiological mechanisms in order to suggest new treatment regimes., (© 2013.)

This article presents a new Dutch multicenter study ("PKU-COBESO") into cognitive and behavioral sequelae of early and continuously treated Phenylketonuria (PKU) patients. Part of the study sample will consist of young adult PKU patients who have participated in a large neuropsychological study approximately 10 years ago, when they were 7-to-15-year-olds (Huijbregts et al., 2002 [1]). Their neurocognitive development will be mapped in association with their earlier and continued metabolic history, taking into account possible changes in, for instance, medication. A second part of the sample will consist of PKU patients between the ages of 7 and approximately 40 years (i.e., born in or after 1974, when neonatal screening was introduced in The Netherlands), who have not participated in the earlier neuropsychological study. Again, their cognitive functioning will be related to their metabolic history. With respect to aspects of cognition, there will be an emphasis on executive functioning. The concept of executive functioning will however be extended with further emphasis on the impact of cognitive deficits on the daily lives of PKU patients, aspects of social cognition, social functioning, and behavior/mental health (i.e., COgnition, BEhavior, SOcial functioning: COBESO). In addition to a description of the PKU-COBESO study, some preliminary results with respect to mental health and social functioning will be presented in this article. Thirty adult PKU patients (mean age 27.8, SD 6.4) and 23 PKU patients under the age of 18 years (mean age 11.0, SD 3.3) were compared to 14 (mean age 26.9 years, SD 5.9) and 7 matched controls (mean age 10.5, SD 2.6) respectively, with respect to their scores on the Adult Self-Report or Child Behavior Checklist (measuring mental health problems) and the Social Skills Checklist or Social Skills Rating System (measuring social skills). Whereas there were very few significant group differences (except for mental health problems in the internalizing spectrum for adult PKU patients), possibly due to the small control groups, several significant associations between mental health problems and Phe levels were observed for the PKU patients. Childhood Phe levels and internalizing problems for adult PKU patients were related; concurrent Phe was associated with both internalizing and externalizing behavioral problems for those under the age of 18. These preliminary results underline the importance of early dietary adherence., (© 2013.)

Background: Our laboratory seeks a pharmacotherapeutic intervention for PKU that utilizes non-physiological amino acids (NPAAs) to block the accumulation of phenylalanine (Phe) in the brain. In previous studies (Vogel et al. 2013), methylation of the amino group of 2-aminoisobutyrate (AIB) provided an enhanced degree of selectivity for Phe restriction into the brain of Pah(enu2) mice in comparison to unmethylated AIB, leading to the hypothesis that 2-(methylamino)alkanoic acid analogs of AIB might represent targeted inhibitors of Phe accretion into the brain., Methods: Pah(enu2) and control mice were intraperitoneally administered (500-750 mg/kg body weight, once daily; standard 19% protein diet) AIB, methyl AIB (MAIB), isovaline, and two MAIB analogs, 2-methyl-2-(methylamino)butanoic (MeVal) and 3-methyl-2-(methylamino)pentanoic (MePent) acids for one week, followed by brain and blood isolation for amino acid analyses using UPLC., Results: In the brain, AIB significantly reduced Phe accretion in Pah(enu2) mice, while MeVal significantly improved glutamine and aspartic acids. Four of five test compounds improved brain threonine and arginine levels. AIB, MAIB and IsoVal significantly reduced blood Phe, with no effect of any drug intervention on other sera amino acids., Conclusions: Further evaluation of AIB and the 2-(methylamino)alkanoic acids as inhibitors of brain Phe accumulation in Pah(enu2) mice is warranted, with more detailed evaluations of route of administration, combinatorial intervention, and detailed toxicity studies., (© 2013.)

Objective: There are several problems associated to the management of patients with phenylketonuria (PKU). Social status could be one of the affecting factors on dietary adherence in these patients. The aim of this study was to evaluate family social status and dietary adherence of PKU patients in Iranian population., Methods: In a cross-sectional study, we studied 105 Iranian PKU patients (born 1984 to 2010), treated and followed at Mofid Children's Hospital, Tehran. Social status was defined by number of children in family, number of affected children in family, maternal and paternal education, marital and employment status of the parents. Age at diagnosis and duration of treatment were also recorded. Mean plasma phenylalanine level was considered as a sign of dietary adherence in PKU patients and was calculated considering the phenylalanine measurements throughout at least one year., Findings: Mean plasma phenylalanine concentration was 5.9±3.6 mg/dl in patients <12 years old and 13.1±3.9 mg/dl in patients >12 years old. Blood phenylalanine concentrations in 47.6% of patients were in normal age-related reference range. There was a significant association between divorced and unemployed parents, and higher levels of blood phenylalanine concentration (P=0.02 and P=0.03 respectively). There was a significant positive correlation between number of affected children in the family (r=0.43, P<0.001), age at diagnosis (r=0.2, P=0.03), treatment duration (r=0.7, P=<0.001) and blood phenylalanine concentrations. There was no significant relation between parental education, family size and dietary adherence., Conclusion: Social status affects dietary adherence to some extent. We suggest exploring care-givers dietary knowledge as the next step to improve dietary compliance in these patients.

Objective: Phenylalanine hydroxylase or its cofactor, tetrahydrobiopterin (BH(4)), deficiency causes accumulation of phenylalanine in body fluids and central nervous system. Considering the fact that hyperphenylalaninemia is a preventable cause of mental retardation in infants, the objective of this study was to determine the incidence of congenital hyperphenylalaninemia in Fars province, south of Iran., Methods: In a period of one year from November 2007 to November 2008 blood samples were withdrawn from all newborns born in Fars province for measurement of serum phenylalanine. The samples with a serum level of≥ 2 mg/dl were referred to pediatric endocrine clinic for confirmation and determination of the type of hyperphenylalaninemia by quantitive serum phenylalanine measurements by using High-Pressure liquid chromatography (HPLC) method., Findings: Nine out of 76966 newborns had a serum phenylalanine level≥2mg/dl, of which 8 cases were confirmed by HPLC. The incidence of the disease was 1:10000. The incidence of mild hyperphenylalaninemia and phenylketonuria (PKU) among the patients was 62.5% and 37.5% respectively and the incidence of BH(4) deficiency was 1/76966., Conclusion: These findings indicate a high incidence of hyperphenylalaninemia, in the newborns from Fars province. The high incidence makes a comprehensive screening program for management of the disease necessary.