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Competing Interests: Declaration of competing interest None.

Phenylketonuria (PKU) is a genetic disorder caused by variations in the phenylalanine hydroxylase (PAH) gene. Among the 3369 reported PAH variants, 33.7% are missense alterations. Unfortunately, 30% of these missense variants are classified as variants of unknown significance (VUS), posing challenges for genetic risk assessment. In our study, we focused on analyzing 836 missense PAH variants following the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines specified by ClinGen PAH Variant Curation Expert Panel (VCEP) criteria. We utilized and compared variant annotator tools like Franklin and Varsome, conducted 3D structural analysis of PAH, and examined active and regulatory site hotspots. In addition, we assessed potential splicing effect of apparent missense variants. By evaluating phenotype data from 22962 PKU patients, our aim was to reassess the pathogenicity of missense variants. Our comprehensive approach successfully reclassified 309 VUSs out of 836 missense variants as likely pathogenic or pathogenic (37%), upgraded 370 likely pathogenic variants to pathogenic, and reclassified one previously considered likely benign variant as likely pathogenic. Phenotypic information was available for 636 missense variants, with 441 undergoing 3D structural analysis and active site hotspot identification for 180 variants. After our analysis, only 6% of missense variants were classified as VUSs, and three of them (c.23A>C/p.Asn8Thr, c.59_60delinsCC/p.Gln20Pro, and c.278A >T/p.Asn93Ile) may be influenced by abnormal splicing. Moreover, a pathogenic variant (c.168G>T/p.Glu56Asp) was identified to have a risk exceeding 98% for modifications of the consensus splice site, with high scores indicating a donor loss of 0.94. The integration of ACMG/AMP guidelines with in silico structural analysis and phenotypic data significantly reduced the number of missense VUSs, providing a strong basis for genetic counseling and emphasizing the importance of metabolic phenotype information in variant curation. This study also sheds light on the current landscape of PAH variants., Competing Interests: Declaration of competing interest No author has any competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study is to present a series of case studies on the real-life use of pegvaliase in Italy in managing patients affected by phenylketonuria (PKU) and provide practical insight and support to healthcare professionals currently approaching and facing this novel enzyme substitution therapy., Methods: A panel of 11 PKU experts from seven leading Italian treatment centers attended online virtual meetings with the aim of reviewing their clinical and practical experiences with pegvaliase based on occurred cases. In selecting the cases, specific consideration was given to the nationwide representation of the centers involved and to the number of patients with PKU managed. Cases were thoroughly reviewed, with comprehensive discussions enabling the identification of key take-home messages regarding pegvaliase therapy., Results: The panel discussed 18 cases, 11 males and 7 females (age range 17-43 years). At the last follow-up (up to 111 weeks after pegvaliase initiation), 11 out of 18 patients (61%) reached Phe levels below 600 μmol/l. Outcomes varied significantly across cases. All cases underscore the potential of pegvaliase in reducing Phe levels, enhancing the quality of life, and promoting social skills and independence. Additionally, the cases highlight the challenges associated with pegvaliase therapy, including managing adverse events and ensuring patient motivation and adherence., Conclusion: This is the first report about the Italian experience of managing patients affected by PKU with pegvaliase. Given the limited real-world data on the use of pegvaliase in PKU management, this case series offers valuable insights into the practical implementation and management of pegvaliase therapy in this Country. Continued research and data collection will be crucial to confirm and progress with this treatment. Despite potential challenges, pegvaliase therapy represents a substantial promise in managing PKU in Italy. Patient education, personalized treatment approaches, and careful monitoring are important to ensure optimal patient outcomes., Competing Interests: Declaration of competing interest None, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Adults with PKU have difficulty maintaining plasma phenylalanine (Phe) in the range that is safe for neurologic function. Elevated plasma Phe is a risk factor for congenital anomalies and developmental delay in offspring resulting from pregnancies with poor Phe control in women with PKU. Enzyme supplementation with pegvaliase allows adults with PKU to eat an unrestricted diet and have plasma Phe levels in a safe range for pregnancy but pegvaliase has not been approved for use in pregnant females with PKU. We report the results of chart review of 14 living offspring of females affected with PKU who were responsive to pegvaliase and chose to remain on pegvaliase throughout their pregnancy., Methods: Fourteen pregnancies (one triplet pregnancy) and their offspring were identified at eight PKU treatment centers and medical records from pregnancy and birth were submitted for this study. Institutional Review Board approval was obtained. Responses to a dataset were provided to a single center and analyzed., Results: Six females and eight males were born without congenital anomalies and all offspring had normal growth parameters. While mothers had preexisting comorbidities, no additional comorbidities were reported in the offspring. Four of eleven infants (excluding triplet pregnancies) were delivered preterm (36%), a higher rate than the general population (12%). A single first trimester (eight weeks) miscarriage in a 40y was not counted in this cohort of 14 live born infants., Conclusion: This retrospective study suggests that pegvaliase is effective at maintaining safe maternal blood Phe levels during pregnancy without deleterious effects on mother or child. A tendency toward premature birth (4/11; 36%) is higher than expected., Competing Interests: Declaration of competing interest None, (Copyright © 2023. Published by Elsevier Inc.)

Background: Pegvaliase, an enzyme substitution therapy, is a treatment option for phenylketonuria (PKU). Due to the neuropathophysiology and disease burden of PKU, individuals can experience baseline anxiety unrelated to pegvaliase therapy. In addition, there are aspects of pegvaliase therapy that may be anxiety-inducing for those considering or receiving treatment. The aim of this manuscript is to present best practice recommendations for the identification and management of anxiety symptoms that can occur along the pegvaliase journey., Methods: A modified Delphi approach was used to seek consensus among a multidisciplinary panel of experts. To this end, an in-person meeting was held that was preceded by a medical specialist- and patient-specific survey to develop preliminary recommendations on ways to address anxiety along the pegvaliase journey. After the meeting, an additional survey was conducted to rank the proposed solutions and mitigation strategies from which a set of recommendations was developed. All recommendations were voted on with the aim of consensus generation, defined as achieving ≥75% agreement among experts., Results: The panel reached consensus on a total of 28 best practice recommendations for the management of anxiety during the pre-treatment, induction and titration, early maintenance (pre-efficacy), and late maintenance (post-efficacy) stages. The recommendations offer strategies to identify and address the most common causes of pegvaliase-related anxiety, including self-injection, side effects, the titration schedule, prescribed dietary changes, and variable time to efficacy. Overall, managing anxiety in those considering or receiving pegvaliase involves patient-centered communication, shared decision-making, and personalized treatment plans., Conclusions: The best practice recommendations described herein can guide healthcare providers in proactively addressing anxiety during the different stages of pegvaliase treatment, and support providers with initiating and managing pegvaliase in individuals who may experience baseline and treatment-related anxiety., Competing Interests: Declaration of Competing Interest The expert panel was compensated for their participation in the in-person advisory board. In addition, KJB received consulting payments from BioMarin, Denali, Chiesi, and Sanofi and speaker fees as well as travel support from BioMarin, Chiesi, and Sanofi; CE received speaker fees from BioMarin; EE received consulting payments and speaker fees from BioMarin. SH received consulting payments from BioMarin, Cycle Therapeutics, PTC Therapeutics, Sanofi, and Vitaflo, speaker fees from Vitaflo, and participated as clinical trial investigator for BioMarin and PTC Therapeutics; BMH received consulting payments from BioMarin, Horizon Therapeutics, and Synlogic; MM received consulting payments from Horizon Therapeutics, BioMarin, Eton Pharmaceuticals, Acer Therapeutics, Ultragenyx, Applied Therapeutics, Jnana Therapeutics, BridgeBio, and Alexion, and participated as clinical trial investigator for Aeglea Biotherapeutics, Reneo Pharmaceuticals, PTC Therapeutics, Homology Medicines, Horizon Therapeutics, Arcturus Therapeutics, Jnana Therapeutics, Synlogic Therapeutics, and BioMarin; SM received consulting payments from Horizon Therapeutics; HN is a clinical trial investigator for BioMarin, Synlogic Therapeutics, Jnana Therapeutics, Sanofi, and PTC Therapeutics and received consulting fees from BioMarin, Synlogic Therapeutics, Jnana Therapeutics, and PTC as well as speakers fees from BioMarin; SS received consulting payments from BioMarin and travel support from BioMarin and NPKUA; LW received consulting payments from BioMarin and Cycle Pharmaceuticals; DAB received consulting payments from Synlogic Therapeutics, Encoded Therapeutics, and Taysha Gene Therapies as well as consulting payments and travel support from BioMarin. MR and MS have no conflicts of interest to declare. KL, SR, and BW are employees and shareholders of BioMarin Pharmaceutical Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In 2019, pegvaliase was approved in Europe for the treatment of phenylketonuria (PKU) in patients aged 16 years and older with blood phenylalanine (Phe) concentrations above 600 μmol/L despite prior management with available treatment options. Since its European approval, German metabolic centres have gained valuable experience, which may be of benefit to other treatment centres managing patients on pegvaliase., Methods: After a virtual meeting that was attended by nine German physicians, three German dietitians and one American physician, a follow-up discussion was held via an online platform to develop a set of recommendations on the use of pegvaliase in Germany. Eight German physicians contributed to the follow-up discussion and subsequent consensus voting, using a modified Delphi technique. The recommendations were supported by literature and retrospectively collected patient data., Results: Consensus (≥75% agreement) was achieved on 25 recommendations, covering seven topics deemed relevant by the expert panel when considering pegvaliase an option for the treatment of patients with PKU. In addition to the recommendations, a retrospective chart review was conducted in seven of the centres and included 71 patients who initiated treatment with pegvaliase. Twenty-seven patients had been treated for at least 24 months and 23 (85.2%) had achieved blood Phe ≤600 μmol/L with some degree of diet normalisation. Of these patients, 14 had physiological blood Phe on a normalised diet., Conclusion: The practical consensus recommendations provide guidance on the different steps along the pegvaliase journey from clinical site requirements to treatment goals and outcomes. The recommendations are intended to support less experienced European metabolic centres with the implementation of pegvaliase, emphasising that a core treatment team consisting of at least a dietitian and metabolic physician is sufficient to initiate pegvaliase and support patients during their treatment journey., Competing Interests: Declaration of Competing Interest JK received consulting payments and speaker fees from Amicus, BioMarin and Sanofi, as well as speakers fees from Shire and research grants from Danone. CB received consulting payments from BioMarin, participated as a clinical trial investigator for BioMarin and received speaker fees from Sanofi-Genzyme, Sobi and Vitaflo. CH and AR are employees of BioMarin Deutschland GmbH. CK received consulting payments from BioMarin. KGP received consulting payments from BioMarin and research fees as well as speaker fees from Vitaflo. FR received consulting payments from BioMarin, Elastrin Therapeutics and Inozyme Pharma, research grants and patents from Inozyme Pharma, speaker fees from BioMarin, Inozyme Pharma and Recordati Rare Diseases and participated as a clinical trial investigator for Aeglea BioTherapeutics, BioMarin, Inozyme Pharma and PTC Therapeutics. SS received consulting payments from BioMarin, Leadiant GmbH and Vertex, speaker fees and travel support from Sobi, and participated as clinical trial investigator for BioMarin. NW received consulting payments from BioMarin, Sanofi and Takeda, speaker fees from Sanofi and Takeda, travel support from Nutricia and Takeda and participated as a clinical trial investigator for BioMarin. ACM received consulting payment from Atheneum, Nestlé and PTC Therapeutics, speaker fees from AIM-PHAMRA Ltd., APR and Nutricia, travel support from Nutricia and participated as a clinical trial investigator for BioMarin and PTC Therapeutics., (Copyright © 2023 BioMarin Deutschland GmbH and BioMarin Pharmaceutical Inc. Published by Elsevier Inc. All rights reserved.)

Phenylketonuria (PKU) is a metabolic disease where Phenylalanine (Phe) rises much above normal levels. Cross-sectional and correlational studies provide valuable information on the importance of maintaining low blood-Phe to achieve good outcomes, but they may be confounded, at least partially, by differences in participant demographics. Moreover, the effect of Phe at older ages is difficult to ascertain because of strong associations between Phe levels across ages. Within-participant studies avoid confounding issues. We have reviewed these studies. We followed PRISMA guidelines to search the literature for studies reporting the impact of Phe changes within participants. Phe was either increased or decreased through diet relaxation/resumption or through pharmacological interventions. Forty-six separate articles reported, singly or in combination, results on cognition (N = 37), well-being (N = 22) and neurophysiological health (N = 14). For all studies, we established, in a binary way, whether a benefit of lower Phe was or was not demonstrated and compared numbers showing benefit versus a null or negative outcome. We then analyzed whether critical parameters (e.g., length of the study/condition for the change, size of Phe change achieved) influenced presence or absence of benefit. For a subset of studies that reported quantitative cognitive outcomes, we carried out a meta-analysis to estimate the size of change in cognitive performance associated with a change in Phe and its significance. There were significantly more studies with benefits than no benefits, both for cognitive and well-being outcomes, and a trend in this direction for neurophysiological outcomes. The meta-analysis showed a highly significant effect size both overall (0.55) and when studies with adults/adolescents were considered separately (0.57). There was some indication that benefits were easier to demonstrate when differences in Phe were larger and achieved across a longer period, but these effects were not always consistent. These results reinforce results from the literature by demonstrating the importance of lower Phe in children as well as in adolescents and adults, even when confounding factors in group composition are eliminated. The field would benefit from further studies where Phe levels are contrasted within-participants to ascertain how much Phe needs to be changed and for how long to see a difference and which measures demonstrate a difference (e.g., which cognitive tasks)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Novel pharmaceutical therapies such as pegvaliase, phenylalanine ammonia lyase (PAL), have enhanced disease control for many individuals with phenylketonuria (PKU). We present a retrospective chart review to assess pegvaliase doses over time in individuals followed at the Boston Children's Hospital PAL Clinic, including those who started pegvaliase in a clinical trial ("trial patients") and those who started after drug came to market ("post-market patients"). Trial patients were on pegvaliase an average of 4.8 years longer, and their mean current pegvaliase dose was 126 ± 92 compared to 223 ± 147 mg/week for post-market patients (p = 0.0155), suggesting that the pegvaliase dose for target efficacy may decrease over time in adults with PKU. In post-market patients, we demonstrated a significant, inverse correlation with dose change and number of weeks from response (r = -0.46, p = 0.046). The entire cohort showed significant variability in terms of time to achieve a therapeutic response, response dose, and current dose. Our data suggest that patients tolerate a reduction in pegvaliase dose over time while maintaining efficacy. This is a clinically meaningful finding as it indicates that patients may reduce number of weekly injections over time on pegvaliase., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Early treated patients with phenylketonuria (PKU) often become lost to follow-up from adolescence onwards due to the historical focus of PKU care on the pediatric population and lack of programs facilitating the transition to adulthood. As a result, evidence on the management of adolescents and young adults with PKU is limited., Methods: Two meetings were held with a multidisciplinary international panel of 25 experts in PKU and comorbidities frequently experienced by patients with PKU. Based on the outcomes of the first meeting, a set of statements were developed. During the second meeting, these statements were voted on for consensus generation (≥70% agreement), using a modified Delphi approach., Results: A total of 37 consensus recommendations were developed across five areas that were deemed important in the management of adolescents and young adults with PKU: (1) general physical health, (2) mental health and neurocognitive functioning, (3) blood Phe target range, (4) PKU-specific challenges, and (5) transition to adult care. The consensus recommendations reflect the personal opinions and experiences from the participating experts supported with evidence when available. Overall, clinicians managing adolescents and young adults with PKU should be aware of the wide variety of PKU-associated comorbidities, initiating screening at an early age. In addition, management of adolescents/young adults should be a joint effort between the patient, clinical center, and parents/caregivers supporting adolescents with gradually gaining independent control of their disease during the transition to adulthood., Conclusions: A multidisciplinary international group of experts used a modified Delphi approach to develop a set of consensus recommendations with the aim of providing guidance and offering tools to clinics to aid with supporting adolescents and young adults with PKU., Competing Interests: Declaration of competing interest The content of this manuscript was based on preparatory pre-meeting activities and presentations and discussions during two advisory board meetings that were coordinated and funded by BioMarin Pharmaceutical Inc. All authors or their institutions received funding from BioMarin to attend at least one or both meetings. Additional disclosures: BKB received consulting payments from BioMarin, Shire, Genzyme, Alexion, Horizon Therapeutics, Denali Therapeutics, JCR Pharma, Moderna, Aeglea BioTherapeutics, SIO Gene Therapies, Taysha Gene Therapy, Ultragenyx, and Inventiva Pharma, participated as clinical trial investigator for BioMarin, Shire, Denali Therapeutics, Homology Medicines, Ultragenyx, and Moderna as well as received speaker fees from BioMarin, Shire, Genzyme, and Horizon Therapeutics. AH received consulting payments from BioMarin, Chiesi, Shire, Genzyme, Amicus, and Ultragenyx, participated as clinical trial investigator for Ultragenyx as well as received speaker fees from Alexion, Amicus, BioMarin, Genzyme, Nutricia, Sobi, and Takeda. ABQ received consulting payments from BioMarin, speaker fees from BioMarin, Nutricia, Vitaflo, Sanofi, Takeda, Recordati, and travel support from Vitaflo. SEC received consulting payments and speaker fees from BioMarin as well as consulting payments from Synlogic Therapeutics. COH was clinical trial investigator for BioMarin and received consulting and speaker payments from BioMarin. SCJH received consulting payments and travel support from BioMarin and Homology Medicines. NL received consulting payments from Alnylam, Amicus, Astellas, BioMarin, BridgeBio, Chiesi, Genzyme/Sanofi, HemoShear, Horizon Therapeutics, Jaguar, Moderna, Nestle, PTC Therapeutics, Reneo, Shire, Synlogic, and Ultragenyx, participated as clinical trial investigator for Aeglea, Amicus, Astellas, BioMarin, Genzyme/Sanofi, Homology, Horizon, Moderna, Pfizer, Protalix, PTC Therapeutics, Reneo, Retrophin/Travere therapeutics, Shire, and Ultragenyx, as well as received speaker fees from Cycle Pharmaceuticals, Leadiant and Recordati. MCM II received consulting payments from BioMarin, Horizon Therapeutics, Rhythm Pharmaceuticals, Applied Therapeutics, Cycle Therapeutics, and Ultragenyx. ALSP received speaker fees from BioMarin. JCR received consulting payments from Applied Pharma Research, Merck Serono, BioMarin, Vitaflo, and Nutricia, speaker fees from Applied Pharma Research, Merck Serono, BioMarin Pharmaceutical, Vitaflo, Cambrooke, PIAM, LifeDiet, and Nutricia, as well as travel support from Applied Pharma Research, Merck Serono, BioMarin, Vitaflo, Cambrooke, PIAM, and Nutricia. SS received consulting payments, research grants, speaker fees, and travel support from BioMarin and participated as clinical trials investigator for BioMarin. ASV received consulting payments from BioMarin, Horizon Therapeutics, and Ultragenyx and participated as clinical trial investigator for Acadia, Alexion, BioMarin, Genzyme, Homology Medicines, Kaleido, Mallinckrodt, and Ultragenyx. JV received consulting payments from BioMarin, LogicBio Pharmaceuticals, Sangamo Therapeutics, Orphan Labs, Synlogic Therapeutics, Sanofi, Axcella Health, Agios Pharmaceuticals, and Applied Therapeutics as well as travel grants from BioMarin and LogicBio Pharmaceuticals. MW received consulting payments, speaker fees, and travel support from BioMarin, and participated as clinical trial investigator for Mallinckrodt, Roche, Wave, Cycle Therapeutics, and Intrabio. ACM participated in strategic advisory boards and received honoraria as a consultant and as a speaker for Merck Serono, BioMarin, Nestlé Health Science (SHS), Applied Pharma Research, Actelion, Retrophin, Censa, PTC Therapeutics, and Arla Food., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

More than 1280 variants in the phenylalanine hydroxylase (PAH) gene are responsible for a broad spectrum of phenylketonuria (PKU) phenotypes. While the genotype-phenotype correlation is reaching 88%, for some inconsistent phenotypes with the same genotype additional factors like tetrahydrobiopterin (BH 4 ), the PAH co-chaperone DNAJC12, phosphorylation of the PAH residues or epigenetic factors may play an important role. Very recently an additional player, the long non-coding RNA (lncRNA) transcript HULC, was described to regulate PAH activity and enhance residual enzyme activity of some PAH variants (e.g., the most common p.R408W) by using HULC mimics. In this review we present an overview of the lncRNA function and in particular the interplay of the HUCL transcript with the PAH and discuss potential applications for the future treatment of some PKU patients., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pegvaliase (Palynziq®) is an enzyme substitution therapy using PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL) to reduce blood phenylalanine (Phe) levels in adults with phenylketonuria (PKU). In Phase 3 clinical studies, all subjects treated with pegvaliase developed anti-drug antibodies. To specifically evaluate pegvaliase-neutralizing antibodies (NAbs) and assess impact on pegvaliase efficacy, a novel hybrid ligand-binding/tandem mass spectrometry NAb assay was developed. Analysis of Phase 3 study samples revealed that pegvaliase NAb titers developed during early treatment (≤6 months after treatment initiation), and then plateaued and persisted in the majority of subjects during late treatment (>6 months). Subjects with the lowest/undetectable NAb titers had relatively high plasma pegvaliase concentrations and experienced the most rapid decline in blood Phe concentrations at relatively low pegvaliase dose concentrations. In contrast, subjects with higher NAb titers generally had lower plasma pegvaliase concentrations on similar low doses, with little change in blood Phe concentrations. However, with additional time on treatment and individualized dose titration, the majority of subjects achieved substantial and sustained blood Phe reduction, including those with higher NAb titers. Moreover, after maturation of the anti-pegvaliase immune response, NAb titers were stable over time and did not rise in response to dose increases; thus, subjects did not require additional dose increases to maintain reduction in blood Phe., Competing Interests: Declaration of Competing Interest All authors were employees and stockholders of BioMarin at the time this work was performed., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: High rates of lost to follow-up (LTFU) adult patients are a major concern in the long-term management of phenylketonuria (PKU). To address this issue, we designed the project "Backtoclinic" with the purpose of identifying LTFU adult PKU patients in Austria as a first step to reestablish appropriate treatment., Subjects and Methods: Individuals born between 1966 and 1999 and diagnosed with PKU through the National Austrian Newborn Screening Program (NANSP) were identified using the NANSP's database. Follow-up data were collected in the Austrian metabolic centers (Medical University of Vienna, Graz, Innsbruck and Salzburg). Patients with no contact to any of these centers within the previous two years were classified as LTFU. Epidemiological characteristics of the whole study population as well as of LTFU- and currently in follow-up patients were analyzed., Results: Between 1966 and 1999, 281 individuals were diagnosed with PKU through the NANSP. Two patients died in their first year of life and were excluded from the analysis. Of the remaining 279 patients (mean age ± SD: 36.7 ± 9.1 y, 42.7% females), 177 (63.4%) are currently LTFU. The rate of LTFU patients is higher in men than in women (68.1% vs 57.5%), and markedly increases with age in both sexes. The gender gap is greatest in young adults (52.6% vs. 25.0% in the age range 20.0-24.9 y) and declines with age (94.4% vs. 80.0% in the age range > 45.0 y)., Conclusions: We found an alarming rate of 63.4% of LTFU adult PKU patients in Austria, and observed a gender gap in the PKU state of care. Our findings illustrate the urgent need for the metabolic community to identify LTFU adult PKU patients and to develop strategies to reestablish appropriate treatment for men and women with PKU., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier Inc.)

Background: Phenylketonuria (PKU) is an inborn error of phenylalanine (Phe) metabolism. Besides dietary treatment, some patients are responsive to and treated with tetrahydrobiopterin (BH4). Our primary objective was to examine whether the 48-hour BH4 loading test misses BH4-responsive PKU patients. Secondary, we assessed if it would be beneficial to 1) use a cut-off value of 20% Phe reduction instead of commonly used 30%, and 2) extend the loading test to 7 days., Methods: 24 patients with a 20-30% decrease of blood Phe levels during their initial 48-hour BH4 loading test or at least one mutation associated with long-term BH4 responsiveness, were invited to participate. 22 of them underwent the 7-day BH4 loading test. During the BH4 loading test, BH4 was administered orally once daily for 7 days (20 mg/kg/day). Blood samples on filter paper were collected at 13 time points. Potential BH4 responders (≥20% decrease in blood Phe concentrations at ≥1 moment within the first 48 h or ≥30% at ≥1 moment during the entire test) underwent a treatment trial to assess true long-term responsiveness (≥30% decrease of Phe levels compared to baseline and/or ≥50% increase in natural protein tolerance in accordance with the Dutch guidelines before 2017). The duration of the treatment trial varied from 2 to 18 months., Results: Of the 22 patients who completed the 7-day BH4 loading test, 2 were excluded, 8 had negative tests and 12 were considered to be potential BH4 responders. Of these 12 potential BH4-responsive PKU patients, 5 turned out to be false positive, 6 true-responder and 1 was withdrawn., Conclusion: Even though the 48-hour BH4 loading test has proven its efficacy in the past, a full week may be necessary to detect all responders. So, if blood Phe concentrations during the 48-hour BH4 test shows a clear tendency, but not sufficient decrease, a full week (with only measurements each 24 h) could be offered. A threshold of ≥20% decrease within 48 h is not useful for predicting true BH4 responsiveness., Competing Interests: Declaration of Competing Interest FJ van Spronsen is/was a member of scientific advisory boards for various defects in amino acid metabolism that are supported by BioMarin, SoBi, Arla Food Int, APR, Eurocept, Lucane, Nestle-Codexis Alliance, Orphan Europe, Rivim Medical BV, Origin, Agios, Vivet, and Nutricia International, has received research grants from Nutricia/Danone, SoBi, Alexion, and Merck Serono/Biomarin, Coexis, NPKUA, ESPKU, Dutch PKU research Foundation, Tyrosinemia Foundation and honoraria as a consultant and speaker from Merck Serono, Biomarin, Pluvia Biotech, Nutricia/Danone, and MendeliKABS. R.A.F. Evers has received financial support from Biomarin for attending symposia. A.M.J. van Wegberg has received a research grant from Nutricia, honoraria from Biomarin as speaker, and travel grants from Nutricia and Vitaflo. E van Dam was a member of a scientific advisory board of Merck-Serono/Biomarin. MC de Vries, MCH Janssen and MR Heiner-Fokkema declare that they have no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In phenylketonuria (PKU), mutations of the phenylalanine hydroxylase (PAH) gene decrease the ability of PAH to convert phenylalanine (Phe) to tyrosine (Tyr), resulting in Phe accumulation in the blood and brain and disruption of neurotransmitter (NT) biosynthesis and metabolism. The following translational study explored the relationship between pegvaliase-mediated Phe correction in plasma and the NT biosynthesis and metabolism pathway in mice and humans with PKU. Lower plasma Phe levels were associated with normalization of the NT biosynthesis pathway which correlated with an improvement in inattention symptoms in subjects with PKU., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Tetrahydrobiopterin (BH 4 ) is the natural cofactor of aromatic amino acid hydroxylases and essential for degradation of phenylalanine and synthesis of catecholamines and serotonin. It can be synthesized either de novo from GTP or through the salvage pathway from sepiapterin. Sepiapterin, a natural precursor of BH 4 , is a more stable molecule and is transported more efficiently across cellular membranes, thus having potentially significant advantage over BH 4 as a pharmacological agent for diseases associated with BH 4 -deficient conditions. We report the results of a first-in-humans, randomized, double-blind, placebo-controlled, dose-ranging, Phase I clinical trial in 83 healthy volunteers of CNSA-001, a novel formulation of sepiapterin. Single oral doses of 2.5-80 mg/kg CNSA-001 caused dose-related increases in plasma sepiapterin (mean C max 0.58-2.92 ng/mL) and BH 4 (mean C max 57-312 ng/mL). Maximum plasma concentrations were achieved in about 1-2 h (sepiapterin) or about 4 h (BH 4 ) after CNSA-001 oral intake. Increases in plasma BH 4 were substantially larger in absolute terms and on a dose-for-dose basis following treatment with CNSA-001 vs. sapropterin dihydrochloride, a synthetic form of BH 4 . The pharmacokinetics of plasma sepiapterin and BH 4 were similar before and after seven days of repeat daily dosing with CNSA-001 at 5, 20 or 60 mg/kg indicating little or no drug accumulation. Oral administration of CNSA-001 resulted in higher concentrations of sepiapterin in fasted vs. fed subjects, but overall BH 4 plasma exposure following CNSA-001 intake increased by 1.7-1.8-fold in fed subjects. CNSA-001 was well tolerated, with no clear dose-relationship for adverse events (AE), no serious AE and no study discontinuations for AE. These data indicate that CNSA-001 is rapidly and efficiently converted to BH 4 in humans supporting further clinical evaluation of CNSA-001 for the management of PKU, primary BH 4 deficiencies and other diseases associated with deficient BH 4 metabolism., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Phenylketonuria (PKU) is caused by a deficiency in phenylalanine hydroxylase enzyme activity that leads to phenylalanine (Phe) accumulation in the blood and brain. Elevated blood Phe levels are associated with complications in adults, including neurological, psychiatric, and cognitive issues. Even with nutrition and pharmacological management, the majority of adults with PKU do not maintain blood Phe levels at or below guideline recommended levels. Pegvaliase, PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL), converts Phe to trans-cinnamic acid and ammonia, and is an investigational enzyme substitution therapy to lower blood Phe in adults with PKU., Methods: Pegvaliase was administered using an induction, titration, and maintenance dosing regimen in adults with PKU naïve to pegvaliase treatment. Doses were gradually increased until blood Phe ≤ 600 μmol/L was achieved. The maintenance dose was the dose at which participants achieved and sustained blood Phe ≤ 600 μmol/L for at least 4 weeks without dose modification. Analyses were performed for participants who achieved (Group A, n = 11) and did not achieve (Group B, n = 13) maintenance dose during the first 24 weeks of study treatment., Results: Baseline mean blood Phe for Group A and Group B were 1135 μmol/L and 1198 μmol/L, respectively. Mean blood Phe ≤ 600 μmol/L was achieved for Group A by Week 11 (mean blood Phe of 508 ± 483 μmol/L) and for Group B by Week 48 (mean blood Phe of 557 ± 389 μmol/L). The most common adverse events involved hypersensitivity reactions, which were mostly mild to moderate in severity and decreased over time. One participant in Group B had four acute systemic hypersensitivity events of anaphylaxis consistent with clinical National Institute of Allergy and Infectious Disease/Food Allergy and Anaphylaxis Network criteria; all events were non-IgE mediated and resolved without sequelae, with pegvaliase dosing discontinued after the fourth event. The incidence and titers of anti-drug antibodies were generally lower in Group A compared to Group B., Conclusions: Pegvaliase administered with an induction, titration, and maintenance dosing regimen demonstrated substantial efficacy at reducing blood Phe in both Group A and Group B by Week 48, with a manageable safety profile in most participants. Blood Phe reduction due to pegvaliase appears to be related to dose, treatment duration, and individual immune response; given additional time on treatment and dose titration, later Phe responders (Group B) achieved benefit similar to early Phe responders (Group A), with similar long-term safety profiles., (Copyright © 2018. Published by Elsevier Inc.)

Phenylketonuria (PKU) is a genetic inborn error in metabolism that impacts many people globally, with profound individual and societal consequences when left untreated. The journey of phenylalanine ammonia lyase (PAL) from plant enzyme to enzyme substitution therapy for PKU is a fascinating story that illustrates the importance of collaboration between basic scientists and industry in the drug development process. The story begins with the curiosity of plant physiologists about the origin of lignin, a polymer involved in maintaining the rigidity of plants. They learned that the critical element in this synthesis was an intermediary enzyme that deaminates phenylalanine to cinnamic acid and ammonia (later called phenylalanine ammonia lyase or PAL). Recognition of this ability to metabolize phenylalanine led to subsequent consideration of PAL as a treatment for PKU. This was initially attempted as enteral therapy with extracted enzyme, but that showed only minimal efficacy. Crucially, further development of PAL as a therapy for PKU required quantities of enzyme that could only be obtained after successfully cloning the gene, expressing the enzyme in vitro and modifying the protein via PEGylation to enable parenteral administration of this non-mammalian enzyme. Ultimately, PEGylated PAL was developed as an enzyme substitution therapy for PKU now approved under the name "Palynziq." The multidisciplinary academic-industrial partnership engaged throughout this process has been key to the successful pursuit of this therapeutic possibility and serves as a model for the development of future innovative therapies., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: Pegvaliase is a recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL) enzyme under investigation for treatment of adult phenylketonuria (PKU). This manuscript describes results of a randomized discontinuation trial (RDT) designed to evaluate the effects of pegvaliase treatment on blood phenylalanine (Phe) and neuropsychiatric outcomes in adults with PKU., Methods: PRISM-2 is a 4-part, Phase 3 study that enrolled adults with PKU receiving pegvaliase treatment (initiated in a prior Phase 2 or Phase 3 study). The RDT, Part 2 of PRISM-2, was an 8-week trial that evaluated change in blood Phe concentrations, neuropsychiatric and neurocognitive measures, and safety outcomes in PRISM-2 participants who had achieved at least a 20% blood Phe reduction from pre-treatment baseline with pegvaliase treatment. Participants were randomized 2:1 to either continue pegvaliase (20 mg/day or 40 mg/day) or switch to matching placebo., Results: The pooled pegvaliase group enrolled 66 participants and each placebo group enrolled 14 participants. The primary endpoint of change in blood Phe concentration from RDT entry to RDT Week 8 was met with clinically meaningful and statistically significant differences between the pegvaliase and placebo groups. Mean (SD) blood Phe at the beginning of the RDT when all participants were receiving pegvaliase was 563.9 μM (504.6) in the group assigned to the 20 mg/day placebo group (n = 14), 508.2 μM (363.7) in those assigned to the 40 mg/day placebo group (n = 14), and 503.9 μM (520.3) in those assigned to continue pegvaliase treatment (n = 58). At Week 8 of the RDT, the least squares mean change (95% confidence interval) in blood Phe was 949.8 μM (760.4 to 1139.1) for the 20 mg/day placebo group and 664.8 μM (465.5 to 864.1) for the 40 mg/day placebo group in comparison to 26.5 μM (-68.3 to 121.3) for the pooled (20 mg/day and 40 mg/day) pegvaliase group (P < 0.0001 for pooled pegvaliase group vs each placebo group). Adverse events (AEs) were usually lower in the pooled placebo group when compared to the pooled pegvaliase group. The most common AEs for the pooled pegvaliase and pooled placebo groups were arthralgia (13.6% and 10.3%, respectively), headache (12.1% and 24.1%), anxiety (10.6% and 6.9%), fatigue (10.6% and 10.3%), and upper respiratory tract infection (1.5% and 17.2%)., Conclusion: Mean blood Phe reduction was sustained in the pegvaliase group, while placebo groups had mean blood Phe concentration increase toward pre-treatment baseline levels. Results from this study confirmed the efficacy of pegvaliase in maintaining reduced blood Phe concentrations with a manageable safety profile for most participants., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: In 2014, recommendations for the nutrition management of phenylalanine hydroxylase deficiency were published as a companion to the concurrently published American College of Medical Genetics and Genomics guideline for the medical treatment of phenylketonuria (PKU). These were developed primarily from a summary of findings from the PKU scientific review conference sponsored by the National Institutes of Health and Agency for Healthcare Research & Quality along with additional systematic literature review. Since that time, the Genetic Metabolic Dietitians International and the Southeast Regional Newborn Screening and Genetics Collaborative have partnered to create a web-based technology platform for the update and development of nutrition management guidelines for inherited metabolic disorders., Objective: The purpose of this PKU guideline is to establish harmonization in treatment and monitoring, to guide the integration of nutrition therapy in the medical management of PKU, and to improve outcomes (nutritional, cognitive, and developmental) for individuals with PKU in all life stages while reducing associated medical, educational, and social costs., Methods: Six research questions critical to PKU nutrition management were formulated to support guideline development: Review, critical appraisal, and abstraction of peer-reviewed studies and unpublished practice literature, along with expert Delphi survey feedback, nominal group process, and external review from metabolic physicians and dietitians were utilized for development of recommendations relevant to each question. Recommendations address nutrient intake, including updated protein requirements, optimal blood phenylalanine concentrations, nutrition interventions, monitoring parameters specific to life stages, adjunct therapies, and pregnancy and lactation. Recommendations were graded using a rigorous system derived from the Academy of Nutrition and Dietetics., Results and Conclusion: These guidelines, updated utilizing a thorough and systematic approach to literature analysis and national consensus process, are now easily accessible to the global community via the newly developed digital platform. For additional details on specific topics, readers are encouraged to review materials on the online portal: https://GMDI.org/., (Copyright © 2016. Published by Elsevier Inc.)

Background: In phenylketonuria (PKU) patients, the combination of two phenylalanine hydroxylase (PAH) alleles is the main determinant of residual enzyme activity in vivo and in vitro. Inconsistencies in genotype-phenotype correlations have been observed in compound heterozygous patients and a particular combination of two PAH alleles may produce a phenotype that is different from the expected one, possibly due to interallelic complementation., Methods: A dual eukaryotic vector system with two distinct PAH proteins N-terminally fused to different epitope tags was used to investigate the co-expression of PAH alleles reported in patients with inconsistent phenotypes. PAH variant proteins were transiently co-transfected in COS-7 cells. PAH activity was measured by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS), and protein expression was measured by Western blot. Genotypes were compared with predicted PAH activity from the PAH locus-specific database (PAHvdb) and with phenotypes and tetrahydrobiopterin (BH4) responsiveness from more than 10,000 PKU patients (BIOPKU database)., Results: Through the expression and co-expression of 17 variant alleles we demonstrated that interallelic interaction could be both positive and negative. The co-expressions of p.[I65T];[R261Q] (19.5% activity; predicted 43.5%) and p.[I65T];[R408W] (15.0% vs. 26.8% activity) are examples of genotypes with negative interallelic interaction. The co-expressions of p.[E178G];[Q232E] (55.0% vs.36.4%) and p.[P384S];[R408W] (56.1% vs. 40.8%) are examples of positive subunit interactions. Inconsistencies of PAH residual enzyme activity in vitro and of PKU patients' phenotypes were observed as well. The PAH activity of p.[R408W];[A300S] is 18.0% of the wild-type activity; however, 88% of patients with this genotype exhibit mild hyperphenylalaninemias (MHPs)., Conclusion: The co-expression of two distinct PAH variants revealed possible dominance effects (positive or negative) by one of the variants on residual PAH activity as a result of interallelic complementation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

For most inherited metabolic disorders on newborn screening (NBS) panels, prompt, expert confirmation and treatment are critical to optimize clinical outcomes for children with inherited metabolic diseases (IMD). In the Washington Metropolitan Area (WMA), 3 different short-term follow-up (STFU) systems exist for linking infants with positive newborn screens for IMD to appropriate specialty care. We diagrammed the STFU systems for the District of Columbia, Maryland and Virginia and calculated clinically relevant intervals of time between NBS collection and diagnosis/treatment initiation. We also surveyed representatives from 48 other state NBS programs to classify the STFU systems in the rest of the country. We found that in the WMA the STFU system that did not include the IMD specialist at the same time as the primary care provider (PCP) was associated with a longer median collection-to-specialist contact interval for true positive NBS for critical diagnoses (p=0.013). Nationally, 25% of state NBS programs report having a STFU system that does not include the IMD specialist at the same time as the PCP. In conclusion, there is variability among the STFU systems employed by NBS programs in the US which may lead to delays in diagnosis confirmation and treatment. National standards for STFU systems that include early involvement of an IMD specialist for all presumed positive NBS results may decrease the collection-to-specialist contact interval which could improve clinical outcomes in children with IMD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Symptoms of attention deficit-hyperactivity disorder (ADHD), particularly inattention, and impairments in executive functioning have been reported in early and continuously treated children, adolescents, and adults with phenylketonuria (PKU). In addition, higher blood phenylalanine (Phe) levels have been correlated with the presence of ADHD symptoms and executive functioning impairment. The placebo-controlled PKU ASCEND study evaluated the effects of sapropterin therapy on PKU-associated symptoms of ADHD and executive and global functioning in individuals who had a therapeutic blood Phe response to sapropterin therapy. The presence of ADHD inattentive symptoms and executive functioning deficits was confirmed in this large cohort of 206 children and adults with PKU, of whom 118 responded to sapropterin therapy. In the 38 individuals with sapropterin-responsive PKU and ADHD symptoms at baseline, sapropterin therapy resulted in a significant improvement in ADHD inattentive symptoms in the first 4 weeks of treatment, and improvements were maintained throughout the 26 weeks of treatment. Sapropterin was well-tolerated with a favorable safety profile. The improvements in ADHD inattentive symptoms and aspects of executive functioning in response to sapropterin therapy noted in a large cohort of individuals with PKU indicate that these symptoms are potentially reversible when blood Phe levels are reduced., (Copyright © 2014. Published by Elsevier Inc.)

High serum phenylalanine in adult patients with phenylketonuria (PKU) causes neuropsychological and psychosocial problems that can be resolved by phenylalanine-restricted diet. Therefore, PKU patients must continue to adhere to phenylalanine-restricted diet for life, although the optimal serum phenylalanine level in later life has yet to be established. The purpose of this review was to establish the optimal serum phenylalanine level in later life of PKU patients. We evaluated oxidative stress status, nitric oxide metabolism, cholesterol-derived oxysterols, vitamin D and bone status, and magnetic resonance imaging (MRI) in adult PKU patients according to serum phenylalanine level. Oxidative stress increased markedly at serum phenylalanine of 700-800 μmol/L. Serum phenylalanine higher than 700-850 μmol/L correlated with the disturbance of nitric oxide regulatory system. Adult PKU patients had poor vitamin D status and exhibited predominance of bone resorption over bone formation. In the brain, the levels of 24S-hydroxycholesterol, a marker of brain cholesterol elimination, were low at serum phenylalanine levels exceeding 650 μmol/L. MRI studies showed high signal intensity in deep white matter on T2-weighted and FLAIR images of PKU patients with serum phenylalanine greater than 500 μmol/L, with decreased apparent diffusion coefficients. Changes in most parameters covering the entire body organs in adult PKU were almost acceptable below 700-800 μmol/L of phenylalanine level. However, the optimal serum phenylalanine level should be 500 μmol/L or less in later life for the brain to be safe., (© 2013.)

Fluctuations in blood phenylalanine concentrations may be an important determinant of intellectual outcome in patients with early and continuously treated phenylketonuria (PKU). This review evaluates the studies on phenylalanine fluctuations, factors affecting fluctuations, and if stabilizing phenylalanine concentrations affects outcomes, particularly neurocognitive outcome. Electronic literature searches of Embase and PubMed were performed for English-language publications, and the bibliographies of identified publications were also searched. In patients with PKU, phenylalanine concentrations are highest in the morning. Factors that can affect phenylalanine fluctuations include age, diet, timing and dosing of protein substitute and energy intake, dietary adherence, phenylalanine hydroxylase genotype, changes in dietary phenylalanine intake and protein metabolism, illness, and growth rate. Even distribution of phenylalanine-free protein substitute intake throughout 24h may reduce blood phenylalanine fluctuations. Patients responsive to and treated with 6R-tetrahydrobiopterin seem to have less fluctuation in their blood phenylalanine concentrations than controls. An increase in blood phenylalanine concentration may result in increased brain and cerebrospinal fluid phenylalanine concentrations within hours. Although some evidence suggests that stabilization of blood phenylalanine concentrations may have benefits in patients with PKU, more studies are needed to distinguish the effects of blood phenylalanine fluctuations from those of poor metabolic control., (© 2013.)

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.)

Objectives: A controlled long-term study was performed to assess the neurological and neuropsychological performance in adult patients with early-treated phenylketonuria (PKU)., Methods: We investigated 57 patients with early-treated classical PKU aged 19 to 41 years (mean age 31 years) and 46 matched healthy controls, matched for age and socioeconomic status. Patients and controls were assessed for their intelligence quotient (IQ), and attention and information-processing abilities. Magnetic resonance imaging (MRI) of the brain was performed in all patients. Neuropsychological assessments and MRI were repeated at a five-year-follow-up., Results: In the five-year interval IQ, information processing and attention of patients and controls remained constant. At both assessment times the IQ scores were significantly lower in patients compared to controls. Older adult patients (>32 years) showed poorer information processing and attention at both assessment times compared to young adult patients (<32 years) and controls. IQ, information processing and attention showed no correlation to imaging results but were significantly correlated to blood phenylalanine (Phe) levels in patients' childhood and adolescence, and Phe levels had been higher in the adolescent years of older adult patients., Conclusions: Cognitive performance in adult patients with early-treated PKU does not seem to be subject to deterioration observable in a five-year interval. Neuropsychological assessment in adults with PKU revealed neurocognitive impairment particularly in older adult patients. This seems to refer to an early relaxation of diet that was recommended when the older patients were adolescents. Results indicate a benefit of dietary control during adolescence in PKU., (© 2013.)

Dietary treatment may be associated with an increased risk of obesity in phenylketonuria (PKU). The earliest studies describe a tendency for overweight in PKU, but not all recent publications confirm this, although there are an increasing number of studies describing increased obesity in female patients with PKU. There is little data describing the metabolic consequences of obesity in PKU. It is difficult to interpret and compare published results due to variable patient age, differing dietary treatment approaches, poor treatment adherence, inconsistencies in metabolic control achieved, variable criteria used to classify overweight. There is also a lack of comparison with normal population data which is widely variable between countries. Generally in PKU it is unknown if obesity etiology is a result of the underlying condition, a treatment consequence, or an outcome of inadequate metabolic control. Differences in treatment strategies, target ranges for blood phenylalanine concentrations and severity of PKU can alter nutritional intakes and dietary experiences which ultimately modulate the course of overweight development. It is clear further investigation is required. Treating overweight and obesity in the general population is difficult and no studies have described the impact of obesity treatment strategies in PKU. However, the PKU management team has an important role in monitoring nutritional status and preventing overweight and obesity. It is important that PKU treatment attends to the general aspects of nutrition, feeding behavior and exercise in order to prevent the development of overweight in these individuals., (© 2013.)

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.)

Patients with phenylketonuria (PKU) encompass an 'at risk' group for micronutrient imbalances. Optimal nutrient status is challenging particularly when a substantial proportion of nutrient intake is from non-natural sources. In PKU patients following dietary treatment, supplementation with micronutrients is a necessity and vitamins and minerals should either be added to supplement phenylalanine-free l-amino acids or given separately. In this literature review of papers published since 1990, the prevalence of vitamin and mineral deficiency is described, with reference to age of treatment commencement, type of treatment, dietary compliance, and dietary practices. Biological micronutrient inadequacies have been mainly reported for zinc, selenium, iron, vitamin B12 and folate. The aetiology of these results and possible clinical and biological implications are discussed. In PKU there is not a simple relationship between the dietary intake and nutritional status, and there are many independent and interrelated complex factors that should be considered other than quantitative nutritional intake., (© 2013 Elsevier Inc. All rights reserved.)

Background: Phenylketonuria (PKU) is a rare inborn error of metabolism caused by phenylalanine hydroxylase enzyme (PAH) deficiency. Treatment constitutes a strict Phe restricted diet with unpalatable amino acid supplements. Residual PAH activity enhancement with its cofactor tetrahydrobiopterin (BH4) is a novel treatment which increases dietary tolerance in some patients and permits dietary relaxation. Relaxation of diet may improve health related quality of life (HRQoL). This prospective cohort study aims to evaluate HRQoL of patients with PKU and effects of BH4 treatment on HRQoL., Methods: Patients aged 4years and older, diagnosed through newborn screening and early and continuously treated, were recruited from eight metabolic centers. Patients and mothers completed validated generic and chronic health-conditions HRQoL questionnaires (PedsQL, TAAQOL, and DISABKIDS) twice: before and after testing BH4 responsivity. Baseline results were compared to the general population. Data collected after BH4 testing was used to find differences in HRQoL between BH4 unresponsive patients and BH4 responsive patients after one year of treatment with BH4. Also a within patient comparison was performed to find differences in HRQoL before and after treatment with BH4., Results: 69/81 (85%) patients completed the questionnaires before BH4 responsivity testing, and 45/69 (65%) participated again after testing. Overall PKU patients demonstrated normal HRQoL. However, some significant differences were found when compared to the general population. A significantly higher (thus better) score on the PedsQL was reported by children 8-12 years on physical functioning and by children 13-17 years on total and psychosocial functioning. Furthermore, adult patients reported significantly lower (thus worse) scores in the TAAQOL cognitive domain. 10 patients proved to be responsive to BH4 treatment; however improvement in their HRQoL after relaxation of diet could not be demonstrated., (© 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: In the past, overtreatment may have resulted in growth impairment in patients with phenylketonuria., Objective: The paper aims to investigate height and body composition in early treated patients with phenylketonuria who were diagnosed between 1981 and 2008., Design: A cross-sectional study of 89 patients with phenylketonuria and 78 controls aged (mean ± SD, in years) 14.4 ± 6.6 and 15.9 ± 7.1, respectively, was undertaken, including anthropometric and body composition evaluation using bioelectrical impedance. Median Phe concentrations in the last year before study enrollment were used as a measure of metabolic control. Natural protein and amino acid mixture intakes were recorded in patients., Results: No statistically significant differences were found on height z-scores between patients and controls aged less than 19 years (p=0.301), although all patients with classical phenylketonuria revealed negative height z-scores, resulting in a mean ± SD of -0.65 ± 0.41. Among participants aged 19 years or more, median (p25-p75) of height was significantly higher in controls [168.0 cm (159.2-174.8)] than in patients [160.5 cm (151.9-167.5)] (p=0.017). No significant differences were found between patients and controls regarding fat mass, fat free mass, muscular mass, body cell mass index and phase angle., Conclusion: Our results suggest that early and continuously treated patients with phenylketonuria born after 1992 can achieve normal growth and body composition, although the negative height z-score in patients with classical phenylketonuria strengthens the continuous need to optimize the quality of their protein intake., (© 2013 Elsevier Inc. All rights reserved.)

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.)