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• This White Paper offers a neurocognitive assessment platform for clinical trials in PKU. • It describes 5 neuropsychological domains relevant to outcomes in PKU. • Criteria for measures to be included in the Assessment Platform are defined. • Both Performance and Patient Reported Outcome measures are recommended. • Once implemented, this Platform may accelerate approval of new PKU treatments. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • Pegvaliase dose for target efficacy may decrease over time in adults with PKU. • Mean pegvaliase dose was lower for the patient cohort on therapy longer. • Pegvaliase dose inversely correlated with number of weeks on an efficacious dose. • Time to pegvaliase response, response dose, and current dose show wide variability. • Pegvaliase dose reduction helps patients take fewer injections and avoid hypophe. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 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. 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). 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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. 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. 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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract 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. [ABSTRACT FROM AUTHOR]

Abstract 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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 /. [ABSTRACT FROM AUTHOR]

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 ( PAH vdb) and with phenotypes and tetrahydrobiopterin (BH 4 ) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Individuals with phenylketonuria (PKU) treated early and continuously are reported to have psychiatric and executive function impairments. The feasibility of screening for psychiatric distress and executive function impairment in individuals with PKU was tested in 3 separate clinics in North America. Methods: Individuals were offered screening for psychiatric distress using the Pediatric Symptom Checklist, the PSC–Youth Report or the Brief Symptom Inventory and executive function impairment using the Behavior Rating Inventory of Executive Function. Gender, age and blood phenylalanine (Phe) concentrations obtained most recently and during the 2years prior to screening were assessed. Results: More than 90% of patients with PKU accepted the screening for psychiatric distress during their routine clinic visit. The screening took 15–20min. 32% of patients screened positive for psychiatric distress and 19% for executive function impairment. More individuals >18years screened positive for psychiatric distress while a similar number screened positive for executive function impairment across age groups. Lower blood Phe levels correlated with negative screening for psychiatric distress. Patients positive for psychiatric distress had higher (p=0.009) median and most recent blood Phe values (p=0.05). Discussion/conclusions: Routine screening for psychiatric distress of patients with phenylketonuria could be easily implemented in current clinic structures. High incidences of positive screens reinforce the need for regular psychiatric assessments of individuals with PKU. Identification and referral to local mental health providers might help to improve the standard of care for individuals with PKU. [Copyright &y& Elsevier]

Abstract: A BH4 loading test was performed in 36 patients from 34 unrelated families. The patients had 29 different genotypes, and previous data on only eight of them were found in the BIOPKU database. Thirteen patients were classified as classic PKU (35.1%), 14 as mild PKU (37.8%) and 9 as MHP (27.0%). Blood Phe levels were shown to reach a plateau after three full days of increased natural protein ingestion. Measuring the 24-hour blood Phe levels (T−24, T−16, T0) on the fourth day of increased protein ingestion before BH4 administration showed that within 24h Phe on average increased by 2.4% in MHP patients, decreased by 2.7% in mild PKU patients and increased by 9.7% in classic PKU patients (NS for all comparisons); Phe only slightly decreased in responders by 0.2% but increased in non-responders by 7.8% (P>0.05). Altogether, 16 of 36 (44.4%) patients represented by 12 of 29 (41.4%) different genotypes were proven to be BH4 responders, and four (10.8%) were slow-responders. Responders were 6/9 (66.7%) MHP patients, 10/14 (71.4%) mild PKU patients and 0/13 classic PKU patients. Twenty of the 29 (68.9%) genotypes harbored at least one mutation with a known PRA of 10% or more but only 11 (55%) of them were BH4-responsive. Spontaneous reduction of blood Phe levels within 24h on the fourth day of natural protein loading was observed only in mild PKU patients and was shown not to be an important part of the BH4-response. 73.3% of genotypes containing at least one allele with a PRA of at least 30% were found to be BH4 responsive; a PRA of at least 15.5% was needed for the responder genotype in our population. [Copyright &y& Elsevier]

Abstract: Medical foods and dietary supplements are used to treat rare inborn errors of metabolism (IEM) identified through state-based universal newborn screening. These products are regulated under Food and Drug Administration (FDA) food and dietary supplement statutes. The lack of harmony in terminology used to refer to medical foods and dietary supplements and the misuse of words that imply that FDA regulates these products as drugs have led to confusion. These products are expensive and, although they are used for medical treatment of IEM, third-party payer coverage of these products is inconsistent across the United States. Clinicians and families report termination of coverage in late adolescence, failure to cover treatment during pregnancy, coverage for select conditions only, or no coverage. We describe the indications for specific nutritional treatment products for IEM and their regulation, availability, and categorization. We conclude with a discussion of the problems that have contributed to the paradox of identifying individuals with IEM through newborn screening but not guaranteeing that they receive optimal treatment. Throughout the paper, we use the nutritional treatment of phenylketonuria as an example of IEM treatment. [Copyright &y& Elsevier]

Abstract: Until today, the mainstay of phenylketonuria (PKU) treatment is a phenylalanine (Phe)-restricted diet. Strict dietary treatment decreases flexibility and autonomy and still has a major impact on patients and their families. Compliance is often poor, particularly in adolescence. The aim of this study was to investigate the effect of the intake of fruits and vegetables containing Phe less than 100mg/100g (‘simplified diet’), as recommended by WHO for all individuals, instead of classical totally restricted diet on the course and treatment control of the disease in a well-characterized PKU cohort (n =80). All individual blood Phe measurements of each patient (1992–2009) were statistically analyzed before and after diet switch. Epidemiological data, age at diagnosis, PAH mutations, BH4 responsiveness, as well as Phe control measurements and detailed diet information were tabulated in a local database. 62.5% had BH4 loading test and 40% had PAH analysis; 50/80 switched from classical to simplified diet, including 26 classical PKU, 13 moderate PKU, 7 mild PKU and 4 mild hyperphenylalaninemia (HPA). Median Phe levels on a simplified diet did not differ significantly to the median Phe levels on classical diet in all disease groups. Our results indicate that a simplified diet has no negative effect on blood Phe control in patients with hyperphenylalaninemia, independent of severity of the phenotype or the age at diet switch, over the period of 3years. Thus, a simpler approach to dietary treatment of PKU available to all HPA patients is more likely to be accepted and adhered by patients and might also increase quality of life. [Copyright &y& Elsevier]

Abstract: Mutational spectrum of the phenylalanine hydroxylase (PAH) deficiency was investigated in 107 families (90% of the Slovene PKU population). The entire coding region of the PAH gene was analyzed with dHPLC to select the samples where subsequently the automated sequencing analysis was performed. MLPA analysis was performed to identify large deletions, which were later confirmed with long-range PCR. Correlations with patients'' phenotypes and genotype-based predictions of BH4-responsiveness were assessed. Altogether, disease-causing mutations were identified on 209 alleles (detection rate 97.7%). A spectrum of 36 different disease-causing mutations was identified: 20 missense mutations (80% of the alleles), eight splicing mutations (13% of the alleles), one nonsense mutation (0.5% of the alleles), four small deletions with frame shift (6% of the alleles), one small insertion with frame shift (0.5% of the alleles), and two large deletions (2% of the alleles). The most frequent mutation was p.R408W in exon 12, representing 29% of the alleles, which is in concordance with other neighboring and/or Slavic PKU populations. Other common mutations were: p.R158Q, p.A403V, p.P281L and p.E390G, accounting for 9%, 7%, 7% and 7% of the alleles respectively. Five novel mutations were detected: c.43_44insAG, c.56_59+1delACAGG, p.V45A, p.L62P and p.R157S. Large deletion of exon 5 (EX5del955) was found in three patients and a deletion of exon 3 (EX3del4765) in one patient. A spectrum of 64 different genotypes was found, seven of them accounting for over than a third of all families. Among thirteen families with homozygous mutation (13% of the PKU population), 10 had p.R408W, two had p.R158Q and one had p.E390G. Among 107 families, 58 were classified as classic PKU (54.2%), 28 as mild PKU (25.9%) and 21 as MHP (19.6%). Twenty-six different genotypes (40.6%) were predicted to be BH4-responsive, represented by 38 different families (35.5%). [Copyright &y& Elsevier]

Abstract: Purpose: To understand current patient selection, dosing, and response criteria used for sapropterin dihydrochloride (sapropterin, Kuvan®) to treat phenylketonuria (PKU). Methods: Results of a 2010 survey of twenty-nine academic medical centers are reported to describe practice patterns in comparison to results of a survey done in 2008 and to what is reported in the literature. Results/conclusions: In addition to reduction in blood phenylalanine (Phe) levels, clinicians report using broader disease-management approaches when evaluating clinical benefit of sapropterin, including consideration of increased Phe tolerance and behavioral changes. Similar approaches are reported in the literature. [Copyright &y& Elsevier]

Abstract: Background: The etiology of reduced bone mineral density (BMD) in phenylketonuria (PKU) is unknown. Reduced BMD may be inherent to PKU and/or secondary to its dietary treatment. Materials and methods: Lumbar BMD was measured by dual-energy X-ray absorptiometry in 53 early and continuously treated PKU patients (median age 16, range 2–35years). First, Z-scores of BMD were correlated to age group, clinical severity of PKU, mean phenylalanine (Phe) concentration and Phe variation in the year prior to DXA scanning, as well as to blood vitamin, mineral, and alkaline phosphatase concentrations. Second, parameters were compared between subjects with reduced BMD (Z-score<−2 SD) and subjects with normal BMD. Results: BMD was significantly reduced in our cohort (p=0.000). Z-scores of BMD were neither significantly correlated to age group, nor clinical severity of PKU. Both mean Phe concentration and Phe variation in the year prior to DXA scanning did not significantly correlate with Z-scores of BMD. Higher blood calcium concentrations were significantly associated with lower BMD (r2 =−0.485, p=0.004). Other biochemical parameters, including vitamin B12 availability markers, did not show significant correlations with Z-score of BMD. Subjects with reduced BMD had significantly higher blood phosphorus concentrations than subjects with normal BMD (p=0.009). No other significant differences were found between both BMD groups. Conclusion: Reduced BMD in PKU is present from early age onward and does not progress with age. Therefore, BMD deserves attention from early age onward in PKU patients. Our findings are consistent with increased bone turnover in PKU. It remains unclear whether reduced BMD is inherent to PKU and/or secondary to its dietary treatment. [Copyright &y& Elsevier]

Abstract: Background: Residual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patient''s genotype. Activity of in vitro expressed mutant PAH may predict the patient''s phenotype and response to tetrahydrobiopterin (BH4), the cofactor of PAH. Methods: A robust LC–ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q, p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice. Results: The PAH assay was linear for phenylalanine and tyrosine (r2 ≥0.99), with a detection limit of 105nmol/L for Phe and 398nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1mmol/L L-Phe; 200μmol/L BH4) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain. Conclusions: This novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions. [Copyright &y& Elsevier]

Abstract: Sapropterin dihydrochloride, a synthetic tetrahydrobiopterin (BH4), works as a chaperone of phenylalanine hydroxylase (PAH) in phenylketonuria (PKU) to facilitate and stabilize folding of PAH into its most active conformation. No standard pharmacogenetic tests exist to identify responsive genotypes. Previous studies have failed to identify genotypes that consistently predict response; they are weakened by varied: 1) doses; 2) response definitions; 3) duration; 4) phenylalanine (PHE) test times during different protein catabolic states; 5) control of dietary PHE. START (sapropterin therapy actual response test) protocol is a double blind, placebo-controlled, 4-week clinical test that obviates the confounders aforementioned. START results were evaluated for response-genotype correlates and trends in molecular characteristics. Results: Seventy-four patients completed START. Thirty-six patients (48.6%) responded, 55 patients'' genotypes are known, 38 unique genotypes are present. Alleles consistently associated with response include Y414C (8/8 patients, 6 genotypes) and I65T (9/9 patients, 6 genotypes). The p.R408W mutation, in which substitution of straight chain arginine with bulky aromatic amine, tryptophan, at the crux of a strategic hinge site activating folding of PAH, amino acid sequence 408, was strongly associated with non-response (21/29 patients non-responsive, 12/17 genotypes non-responsive). Genotypes containing at least one allele with ≥25% residual activity compared to wild type, were strongly associated with response. Conclusions: The START protocol provides a rigorous pharmacogenetic test to identify sapropterin responsiveness and genotypes associated with responsiveness and non-responsiveness. Some genotypes were found to be predictive of responsiveness or non-responsiveness, and responsiveness was associated with specific alleles. The START protocol provides a reliable test for sapropterin responsiveness and will continue to improve understanding of how PKU mutations impact PAH protein-folding dynamics and enhance understanding of PKU disease and its management. [Copyright &y& Elsevier]

Abstract: In phenylketonuria, knowledge about the relation between behavior and plasma phenylalanine is scarce. The aim of this study was to determine whether high phenylalanine is associated with disturbed behavior noticed by the patient and or close environment (parents or partners). 48 early treated PKU patients (median age 8.5, range 0–35years) participated (median phenylalanine concentration in total sample 277 (range 89–1171) μmol/l; and in patients <12years 238 (range 89–521) μmol/l). After sending blood samples, patients or close environment were interviewed with a standardized questionnaire whether they noticed hyperactivity, annoying behavior, mood swings and introvert or extravert behavior. The interviewer as well as the respondents were blinded with regard to the phenylalanine concentration. Results: Patients reported less deviant behavior compared to close environment. Mood swings were positively associated with phenylalanine concentrations in the total group (P=0.039) and patients <12years (P=0.042). The relationships between temporary high phenylalanine concentrations and hyperactivity, annoying behavior, introvert and extravert behavior were not statistically significant. Conclusion: there is a positive association between phenylalanine concentrations and mood swings. [Copyright &y& Elsevier]

Abstract: Background: The prevalence of phenylalanine hydroxylase (PAH)-deficient phenylketonuria (PKU) in Turkey is high (1 in 6500 births), but data concerning the genotype distribution and impact of the genotype on tetrahydrobiopterin (BH4) therapy are scarce. Objective: To characterize the phenotypic and genotypic variability in the Turkish PKU population and to correlate it with physiological response to BH4 challenge. Methods: We genotyped 588 hyperphenylalaninemic patients and performed a BH4 loading test (20mg/kg bw) in 462 patients. Residual PAH activity of mutant proteins was calculated from available in vitro expression data. Data were tabulated in the BIOPKU database (www.biopku.org). Results: Eighty-eight mutations were observed, the most common missense mutations being the splice variant c.1066-11G>A (24.6%). Twenty novel mutations were detected (11 missense, 4 splice-site, and 5 deletion/insertions). Two mutations were observed in 540/588 patients (91.8%) but in 9 patients atypical genotypes with >2 mutations were found (8 with p.R155H in cis with another variant) and in 19 patients mutations were found in BH4-metabolizing genes. The most common genotype was c.1066-11G>A/c.1066-11G>A (15.5%). Approximately 22% of patients responded to BH4 challenge. A substantial in vitro residual activity (average >25% of the wild-type enzyme) was associated with response to BH4. In homozygous genotypes (n =206), both severity of the phenotype (r =0.83) and residual PAH activity (r =0.85) correlate with BH4 responsiveness. Conclusion: Together with the BH4 challenge, these data enable the genotype-based classification of BH4 responsiveness and document importance of residual PAH activity. This first report of a large-scale genotype assessment in a population of Turkish PKU patients also documents a high prevalence (47%) of the severe classic phenotype. [Copyright &y& Elsevier]

Abstract: PKU is caused by mutations in PAH. A c.30C>G synonymous variation in exon 1, previously reported as neutral, was observed in two patients. The variation creates a GGG triplet, which is part of several exonic splicing silencer (ESS) motifs. Because the 5′-splice site of PAH exon 1 is intrinsically weak and therefore could be responsive to a new flanking ESS, we hypothesized that c.30C>G could cause aberrant mRNA splicing. We demonstrate that c.30C>G causes aberrant mRNA splicing in two different reporter minigenes, and that this is abolished if a preexisting flanking GGG triplet is disrupted. GGG triplets are part of the consensus motif bound by splicing-inhibitory hnRNPH proteins and we observed a dramatic increase in hnRNPH binding to c.30C>G PAH RNA. We conclude that c.30C>G creates a hnRNPH-binding ESS, which can disrupt mRNA splicing. A disease-causing mutation in HEXB, which has previously been associated with exon skipping in patients also creates a GGG triplet. We show that the mutant HEXB motif causes exon skipping of a reporter minigene and that this is also influenced by a flanking GGG triplet. We suggest that aberrant splicing caused by creation/abolishment of GGG triplets located together with a preexisting flanking GGG triplet, may be an underreported cause of human disease. It is important to recognize that exonic sequence changes may disrupt mRNA splicing. This is particularly important in PAH, since PKU patients harboring such mutations are unlikely to respond to therapy with 6R-tetrahydrobiopterin (BH4), despite the fact that the genetic code indicates otherwise. [Copyright &y& Elsevier]

Abstract: Phenylketonuria (PKU) requires a lifelong low-phenylalanine (phe) diet where protein needs are met by consumption of a phe-free amino acid (AA) formula; complaints of persistent hunger are common. Foods made with glycomacropeptide (GMP), an intact protein that contains minimal phe and may promote satiety, provide an alternative to AA formula. The objective was to assess the ability of a GMP breakfast to promote satiety and affect plasma concentrations of AAs, insulin, and the appetite stimulating hormone ghrelin in those with PKU, when compared to an AA-based breakfast. Eleven PKU subjects (8 adults and 3 boys ages 11–14) served as their own controls in an inpatient metabolic study with two 4-day treatments: an AA-based diet followed by a diet replacing all AA formula with GMP foods. Plasma concentrations of AAs, insulin and ghrelin were obtained before and/or 180min after breakfast. Satiety was assessed using a visual analog scale before, immediately after and 150min after breakfast. Postprandial ghrelin concentration was significantly lower (p =0.03) with GMP compared to an AA-based breakfast, with no difference in fasting ghrelin. Lower postprandial ghrelin concentrations were associated with greater feelings of fullness after breakfast suggesting greater satiety with GMP compared to AAs. Postprandial concentrations of insulin and total plasma AAs were higher after a GMP breakfast compared to an AA-based breakfast consistent with slower absorption and less degradation of AAs from GMP. These results show sustained ghrelin suppression, and suggest greater satiety with ingestion of a meal containing GMP compared with AAs. [Copyright &y& Elsevier]

Abstract: Phenylketonuria is discussed from an European perspective, addressing the need of common definitions of terms commonly used, the need of a world-wide guideline on the diagnosis and treatment of phenylketonuria, the differences between existing European guidelines, and day-to-day care, further directives for the near future, and changing the concept from compliance to concordance, in which patients have a more clearly defined responsibility. [Copyright &y& Elsevier]

Abstract: To gain better insight in the most current diagnosis and treatment practices for phenylketonuria (PKU) from a broad group of experts, a European PKU survey was performed. The questionnaire, consisting of 33 questions, was sent to 243 PKU professionals in 165 PKU centers in 23 European countries. The responses were compiled and descriptive analyses were performed. One hundred and one questionnaires were returned by 93/165 centers (56%) from 19/23 European countries (83%). The majority of respondents (77%) managed patients of all age groups and more than 90% of PKU teams included physicians or dieticians/nutritionists. The greatest variability existed especially in the definition of PKU phenotypes, therapeutic blood phenylalanine (Phe) target concentrations, and follow-up practices for PKU patients. The tetrahydrobiopterin (BH4; sapropterin) loading test was performed by 54% of respondents, of which 61% applied a single dose test (20mg/kg over 24h). BH4 was reported as a treatment option by 34%. This survey documents differences in diagnostic and treatment practices for PKU patients in European centers. In particular, recommendations for the treatment decision varied greatly between different European countries. There is an urgent need to pool long-term data in PKU registries in order to generate an evidence-based international guideline. [Copyright &y& Elsevier]

Abstract: Lifelong treatment of phenylketonuria (PKU) includes a phenylalanine (phe) restricted diet that provides sufficient phe for growth and maintenance plus phe-free amino acid formula to meet requirements for protein, energy and micronutrients. Phe tolerance (mgphe/kg body weight/day) is the amount of phe those with PKU can consume and maintain acceptable blood phe levels; it requires individual assessment because of varying phenylalanine hydroxylase activity. The objective was to reassess phe tolerance in eight adults with PKU considering phe requirements, blood phe levels, genotype and phe tolerance at 5years of age. Subjects had not received a personalized assessment of phe tolerance in several years, and five subjects were overweight, body mass index (BMI) 25–28. With the guidance of a metabolic dietitian, seven subjects increased phe tolerance (by 15–173%) without significantly increasing blood phe concentration. Increased phe tolerance was associated with both improved dietary compliance and inadequate phe intake at the onset of the protocol compared with current requirements. Improved dietary compliance reflected increased consumption of protein equivalents from amino acid formula and increased frequency of formula intake, from 2.2 to 3 times per day. Predictors of higher final phe tolerance following reassessment included being male and having a lower BMI (R 2 =0.588). This suggests that the rising trend of overweight and obesity may affect assessment of phe tolerance in adults. Therefore, interaction with the metabolic dietitian to reassess phe tolerance in relation to body mass is essential throughout adulthood to insure adequate intake of phe to support protein synthesis and prevent catabolism. [Copyright &y& Elsevier]

Abstract: Specific mutations in the gene encoding phenylalanine hydroxylase (PAH), located on chromosome 12q22-24.1, are linked to tetrahydrobiopterin (BH4; sapropterin)-responsive phenylketonuria (PKU). Diagnosis is usually done through the newborn screening for PKU, followed by a BH4 loading test. So far, more than 60 mutant alleles, presenting with a substantial residual PAH activity (average ∼47%), were identified in more than 500 patients worldwide. We investigated the predictive value of BH4-responsive PAH mutations in Croatian population. From a group of 127 PKU patients, 62 were selected (based on the genotype) as potentially BH4-responsive and 39 loaded with BH4 (20mg/kg). The overall frequency of BH4-responsiveness (>30% blood phenylalanine reduction within 24h) was 36% (14 out of 39 patients with 23 different genotypes), significantly less than expected. The best responders were patients with mild hyperphenylalaninemia (4/4; 100%), followed by mild PKU (8/9; 89%), and classical PKU (2/26; 8%). The most common BH4-responsive genotypes were p.E390G/p.R408W and p.P281L/p.E390G. These genotypes correspond for approximately >30% residual PAH activity. The p.E390G mutation was 100% associated with BH4-responsiveness, regardless of the second allele (p.R408W, p.P281L, p.F55Lfs, p.L249P). With regard to the predicted relative PAH activity of recombinantly expressed mutant alleles, there was a significant (p <0.002) difference between BH4-responders and non-responders. In a general Croatian PKU population, disease-causing mutations were identified on 226 alleles (99%). There were 35 different mutations: 21 missense, 8 splice site, 3 nonsense, 2 single nucleotide deletions, and 1 in-frame deletion. Four mutations are reported for the first time: p.E76D, p.L333P, p.G346E, and IVS8-2A>G. Five mutations accounted for over two-thirds of investigated alleles: p.L48S, p.R261Q, p.P281L, p.E390G, and p.R408W. Thus, the Croatian PKU population seems to be more homogenous than some other Mediterranean or Central European populations. This study reveals the importance of a full genotype for the prediction of BH4-responsiveness. In contrast to previous assumption and with exception of the p.E390G mutation, single allele mutations are not reliable for the selection of potential PKU candidates for pharmacological therapy with BH4. [Copyright &y& Elsevier]

Abstract: Variability of metabolic control in phenylketonuria (PKU) potentially affects cognitive outcome in early and continuously treated children with this condition. The possibility that homeostasis is more important than the absolute level of exposure to phenylalanine (phe) has not previously been examined. A meta-analysis of 40 studies showed that in children with phenylketonuria (PKU), mean lifetime blood phe levels were significantly correlated with Full Scale IQ (FSIQ) (r =−0.34). A similar correlation (r =−0.35) was found between FSIQ and mean exposure during 0–12 years of age. Most of the studies in the meta-analysis, however, included children who had discontinued the phe restricted diet. None examined the impact of fluctuations in metabolic control in continuously treated children. This is important because new therapies may increase stability in blood phe levels. The question has arisen whether these therapies are beneficial in children whose blood phe levels are generally within the recommended range of 120–360μmol/L. In this study, we describe the relationship between FSIQ and two parameters of metabolic control: (1) mean blood phe level of all reported specimens for each subject, and (2) variability of the blood phe level as indicated by the standard deviation of blood phe levels for each subject. Analyses were performed using lifetime phe levels and levels during three periods (0–6 years, 0–10 years, and >10 years of age). The most recent FSIQ for each child was used in the correlation analyses. Data were collected from medical records on all 46 children born between 1999 and 2006 with early and continuously treated PKU followed at the Metabolism Program at Children’s Hospital Boston. The mean age of the children at the time of their most recent FSIQ test was 7.5+3.3 (2.9–15.5) and their mean FSIQ was 104+15 (68–143). The mean lifetime blood phe level in these children was 312+132μmol/L (125–852). The standard deviation of blood phe levels was 182+72μmol/L (96–336). The correlation between lifetime blood phe levels and most recent FSIQ was −.17 (p =0.38) and the correlation between standard deviation of blood phe levels and most recent FSIQ was −.36 (p =.058), not reaching significance, but indicating a trend. These results indicate that stability of blood phe levels may be more important to cognitive functioning than overall exposure to phe in early and continuously treated PKU. In treating PKU, attention should be given to variability in blood phe levels as well as maintenance of phe levels within the recommended range. [Copyright &y& Elsevier]

Abstract: Hyperhomocysteinemia has occasionally been reported in patients with phenylketonuria (PKU) and B-vitamin deficiency. In our study total homocysteine (tHcy) and B-vitamins were measured in treated PKU patients and healthy controls. In the patients, dietary parameters and genetic polymorphisms affecting the Hcy pathway were investigated to identify parameters modulating tHcy. A case control study including 37 PKU patients and 63 healthy controls was conducted. t-Tests for independent samples were used to test between groups. Multiple regressions with tHcy as dependent variable were calculated. Hardy–Weinberg expectations were tested against the observed distribution of genotypes applying the Chi-square goodness-of-fit method. THcy concentrations were not significantly different (p =0.059) while folate and cobalamin (Cbl) concentrations were significantly higher in PKU patients compared to controls. However, 29.7% of patients had tHcy concentrations >97th centile. THcy did not vary with age nor correlate with folate and Cbl concentrations probably due to high saturatory levels. The presence of genetic polymorphisms had no impact on tHcy. In conclusion, in PKU patients treated with amino acid mixtures enriched with B-vitamins, tHcy is not significantly higher than in healthy controls, but tHcy concentrations exceed the 97th centile in about one third of patients. Even higher B-vitamin saturation may be required to further decrease tHcy concentrations and factors generally influencing tHcy such as betaine are to be investigated in PKU patients in the future. [Copyright &y& Elsevier]

Abstract: We describe the treatment, the clinical, and biochemical findings and the outcome of 26 patients with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency and 10 patients with dihydropteridine reductase (DHPR) deficiency. These are the two most common forms of the autosomal-recessively inherited tetrahydrobiopterin (BH4) deficiency. Time of diagnosis, dosage of BH4 and neurotransmitter precursors, folinic acid substitution, and levels of 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in cerebrospinal fluid (CSF) are essential parameters in the follow-up of patients. Unfortunately, treatment protocols vary greatly among patients and clinical centers, and CSF investigations and outcome assessments are not always available. Seventeen patients with PTPS deficiency and four patients with DHPR deficiency were diagnosed within 2 months after birth. In 14 patients with PTPS deficiency (54%; 9 early and 5 late diagnosed) and 2 patients with DHPR deficiency (20%; all early diagnosed) no developmental delay is observed, while in 10 patients with PTPS deficiency (38%; 6 early and 4 late diagnosed) and 8 patients with DHPR deficiency (80%; 2 early and 6 late diagnosed) development was delayed. Two PTPS-deficient patients died in the newborn period. DHPR deficiency seems to be more severe than PTPS deficiency and it is clearly the onset of treatment that determines the outcome. Our data suggest that diagnosis within the first month of life is essential for a good outcome and that low CSF5 HIAA and HVA values in CSF could be an indicator for the ongoing developmental impairment, even in the absence of neurological symptoms. [Copyright &y& Elsevier]

Abstract: Some individuals with phenylketonuria (PKU) respond to pharmacologic treatment with tetrahydrobiopterin (BH4) by a reduction in the blood phenylalanine concentration. This can result in increased dietary tolerance for phenylalanine or, in rare instances, replacement of the phenylalanine-restricted diet. BH4 is now available as sapropterin dihydrochloride under the name KUVAN, a formulation of natural BH4. This commentary contains recommendations for determining responsiveness to sapropterin dihydrochloride. The recommendations include challenging with an initial daily dose of 20mg/kg and blood phenylalanine determinations pre-challenge and on days 1, 7, and 14 with the option of an additional continuation to day 28 if required to clarify whether a response has occurred. An algorithm depicting this recommendation for the challenge is included. The most widely accepted standard of response is ⩾30% reduction in the blood phenylalanine concentration, but a lower degree of response might also be considered clinically meaningful in some individual circumstances. Issues include the potential treatment of those with mild hyperphenylalaninemia who are not on diet, challenging neonates who have hyperphenylalaninemia identified by newborn screening, and the use of sapropterin dihydrochloride in treatment of maternal PKU pregnancies. These recommendations are intended to provide a basis for the use of sapropterin dihydrochloride in the treatment of PKU but may be altered after close observation of treated patients and carefully performed research. [Copyright &y& Elsevier]

Abstract: Phenylketonuria (PKU, MIM 261600; EC 1.14.16.1) results from mutations in the phenylalanine hydroxylase (PAH) gene. Newborn metabolic disease screening uses blood dried on filter paper (DBS) to prospectively identify candidate newborns affected with PKU via an elevated concentration of phenylalanine. However, it is then important to confirm the specific category of PKU since classical PKU requires a stringent diet while milder categories may not require diet and a very important BH4-responsive category may be treated with the PAH cofactor 6R-tetrahydrobiopterin (BH4). Since there is a close genotype–phenotype correlation in PKU, determining the PAH genotype can be extremely important for therapy as well as prognosis. A simple and rapid method of accurately determining the PAH genotype would be a valuable addition to the diagnosis of PKU. Described herein is a means to identify variants in the PAH gene using high-resolution melt profiling, which compares the thermal denaturation profile of a patient sample to that of a control. Regions where the patient and control samples produce a common profile were not further evaluated, while those regions where the patient profile deviates from the control were assessed by DNA sequencing. Additionally described is a scheme utilizing redundant analysis with melt profile controls and a novel multiplex genotyping assay to triage deviation owing to known polymorphisms. Two mutations were identified in 93 of the 95 patients assessed and in the remaining two patients a single mutation was identified. Melt profiling provided 99% sensitivity to identify sequence variants in the PAH gene. [Copyright &y& Elsevier]

Abstract: Classic phenylketonuria (PKU) is characterized by severe mental retardation in untreated individuals and mild neurocognitive abnormalities in some early treated adults. The exact biochemical mechanisms underlying this neurotoxicity remain undetermined. Several theories implicate abnormal cerebral energy utilization and alterations in biochemical pathways that involve glucose metabolism. This pilot study was undertaken to investigate whether 18F-deoxyglucose positron emission tomography (PET) is an effective tool to study cerebral glucose metabolism in early treated PKU. After PET coregistration with SPGR MRI, relative glucose metabolic rates (rGMR) at the center of standard atlas positions was determined. Repeated measures MANOVA was used to assess regional metabolic differences, which were then correlated with age-specific and day-of-scan plasma phenylalanine and age. Patients with PKU in comparison to controls had decreased rGMR in cortical regions including the prefrontal, somatosensory, and visual cortices, and increased activity in subcortical regions including the striatum and limbic system. Day-of-scan phenylalanine correlated with abnormal activity in subcortical structures, and older age was associated with decreased activity in the prefrontal and visual cortices. The clinical significance of these abnormalities of glucose metabolism in specific areas of the brain remains unknown. [Copyright &y& Elsevier]

Abstract: From all the different molecular mechanisms put forward to explain the basis of BH4 responsiveness in PKU patients, a clear picture is now emerging based on the results from expression studies performed with a number of missense mutations identified in patients with a positive response in BH4 loading tests. Two of the proposed mechanisms, namely decreased binding affinity of the mutant proteins for the natural cofactor and stabilization effect of BH4, have been confirmed for several PKU mutations and the results are reviewed here. The actual view supports a multifactorial basis of the response, highlighting the necessity of detailed in vitro characterization of each mutant PAH protein. Several of the confirmed molecular mechanisms may be operating simultaneously, as exemplified in the data presented, and this may result in different degrees of BH4 responsiveness. [Copyright &y& Elsevier]

Abstract: Patients with tetrahydrobiopterin (BH4)-responsive phenylalanine hydroxylase (PAH) deficiency may benefit from BH4 therapy instead or in addition to the low-phenylalanine diet. Different loading test protocols are currently used to detect these patients. As a consequence, data on the rate of BH4-responsiveness within patients with mild phenylketonuria (PKU) and/or more severe phenotypes show high variation and a more sensitive and standardised BH4 loading test protocol needs to be defined. We modified the current standard BH4 loading test (20mg/kg) to a second administration of 20mg/kg after 24h and extended blood sampling to 48h in 24 patients with PAH deficiency. Using this extended loading test (2×20mg BH4/kg), the rate of BH4-responsiveness was calculated at 8, 24, and 48h after BH4 administration. We defined three groups of patients: “rapid responders” in 10/24 patients (4 mild HPA, 2 mild PKU, 2 moderate PKU, and 2 classic PKU), “moderate responders” in 4/24 patients (4 classic PKU), and “slow responder” in 4/24 patients (4 mild PKU). Six out of 24 patients (1 mild HPA, 1 moderate PKU, and 4 classic PKU) were found to be “non-responder.” Individual phenylalanine profiles show variations in responsiveness at different time points and sampling over 48h was more informative than over 24h in patients with mild and moderate PKU compared to mild HPA. Analysis of BH4 loading tests in 209 patients with the standard BH4 loading test protocol confirms only minor importance of the 24h response: the rate of responsiveness to BH4 after 24h was shown to be equal to or even lower than after 8h among most phenotypes. However, extension of the BH4 loading test to 48h and repeated BH4 administration seems to be useful to detect BH4-responsiveness in more severe phenotypes and allows detecting “slow responders” who may benefit from BH4 therapy. [Copyright &y& Elsevier]

Abstract: Hyperphenylalaninemia caused by phenylalanine hydroxylase (PAH) deficiency requires lifelong rigorous diet starting in early infancy to prevent severe neurodevelopmental handicap. In a considerable number of children with mild hyperphenylalaninemia, long-term tetrahydrobiopterin (BH4) treatment significantly improves phenylalanine (phe) tolerance, but it has never been investigated in classic phenylketonuria (PKU). We performed a BH4-loading test in 40 consecutive infants with phe serum concentrations exceeding 240μM, who had been detected by newborn screening programs. Eighteen out of 40 infants were found to be BH4 responsive. Five of them, responding to the neonatal BH4-loading test, showed a phe tolerance of less than 20mg/kg/day and a phe pretreatment level of >1000μM. They were treated with BH4 (20mg/kg/day) over a period of 24 months. All five children had a sustained response to BH4, allowing substantial easing of dietary restrictions. Before BH4 treatment daily phe tolerance was 18–19mg/kg, increasing to 30–80mg/kg on BH4 treatment and decreasing again to 12–17mg/kg after termination of BH4 treatment. Mutation analysis revealed compound heterozygosity for a putative null and a variant PAH mutation in four patients and homozygosity for a variant PAH mutation in one patient. We conclude that BH4 sensitivitiy is not restricted to mild hyperphenylalaninemia and that long-term BH4 treatment may also improve phenylalanine tolerance in a considerable number of children with a more severe PKU phenotype. [Copyright &y& Elsevier]

Abstract: Tetrahydrobiopterin (BH4) deficiency among newborns with hyperphenylalaninemia must be rapidly diagnosed and distinguished from classical phenylketonuria (PKU) to initiate immediately specific treatment and to prevent irreversible neurological damage. The characteristic pattern of urinary pterins makes it possible to differentiate between PKU and BH4 deficiencies, and to identify different variants of BH4 deficiency. However, collection, storage, and shipment of urine samples for pterin analysis is cumbersome. A method for the measurement of different pterins (neopterin, biopterin, and pterin) in blood collected on filter paper was developed as a potential alternative to the screening for BH4 deficiencies in urine and for the monitoring of BH4 pharmacokinetics. Pterins pattern in blood spots was comparable with those in plasma and urine. We thus established reference values for pterins in blood spots in patients with hyperphenylalaninemia and identified new patients with GTP cyclohydrolase I deficiency, 6-pyruvoyl-tetrahydropterin synthase deficiency, and dihydropteridine reductase deficiency using dried blood spots on filter paper. [Copyright &y& Elsevier]

Abstract: The clinical, nutritional, and neuropsychological data of 11 mild/moderate PKU patients after one year of treatment with BH4 are evaluated. BH4 monotherapy was introduced at 5mg/kg/day in 14 PKU patients. In 11/14 patients, Phe tolerance increased significantly from 356±172 to 1546±192mg/day (p =0.004), and special PKU formula was gradually reduced until complete removal. In them, mean plasma Phe concentrations remained below 360μmol/L at 5mg BH4/kg/day (7mg/kg/day in one patient). BH4 therapy was stopped in three patients (V388M/P362T and R243Q/IVS10-11G>A genotypes) because it was not possible to improve Phe tolerance and to remove formula intake. Serum micronutrients were not significantly different at the start of treatment and at one year follow-up, except for selenium, which increased significantly after one year of therapy (p =0.017). Anthropometric, and nutritional measurements were within the age- and sex-specific percentiles for a healthy population after one year therapy. Neuropsychological follow-up indicated that intelligence scores persisted within normal limits. In terms of patients’ genotype, we confirmed that the P275S mutation combined with R408W was associated with long-term BH4 responsiveness, while the combination of P362T/V388M, and R243Q/IVS10-11G>A resulted in poor metabolic control in long-term BH4 therapy. In summary, our data confirm that BH4 is a safe, and effective therapy in a selected group of mild, and moderate PKU patients who respond to the BH4 loading test. Low doses of BH4 in monotherapy permit withdrawal of the special formula and guarantee a good clinical and nutritional outcome with no adverse side effects in PKU patients. [Copyright &y& Elsevier]