Tetrahydrobiopterin treatment in phenylketonuria:A repurposing approach

In phenylketonuria (PKU) patients, early diagnosis by neonatal screening and immediate institution of a phenylalanine-restricted diet can prevent severe intellectual impairment. Nevertheless, outcome remains suboptimal in some patients asking for additional treatment strategies. Tetrahydrobiopterin (BH4) could be one of those treatment options, as it may not only increase residual phenylalanine hydroxylase activity in BH4-responsive PKU patients, but possibly also directly improves neurocognitive functioning in both BH4-responsive and BH4-unresponsive PKU patients. In the present review, we aim to further define the theoretical working mechanisms by which BH4 might directly influence neurocognitive functioning in PKU having passed the blood-brain barrier. Further research should investigate which of these mechanisms are actually involved, and should contribute to the development of an optimal BH4 treatment regimen to directly improve neurocognitive functioning in PKU. Such possible repurposing approach of BH4 treatment in PKU may improve neuropsychological outcome and mental health in both BH4-responsive and BH4-unresponsive PKU patients

Gut-microbiome composition in response to phenylketonuria depends on dietary phenylalanine in BTBR Pah^enu2 mice

Phenylketonuria (PKU) is a metabolic disorder caused by a hepatic enzyme deficiency causing high blood and brain levels of the amino acid Phenylalanine (Phe), leading to severe cognitive and psychological deficits that can be prevented, but not completely, by dietary treatment. The behavioral outcome of PKU could be affected by the gut-microbiome-brain axis, as diet is one of the major drivers of the gut microbiome composition. Gut-microbiome alterations have been reported in treated patients with PKU, although the question remains whether this is due to PKU, the dietary treatment, or their interaction. We, therefore, examined the effects of dietary Phe restriction on gut-microbiome composition and relationships with behavioral outcome in mice. Male and female BTBR Pah(enu2) mice received either a control diet (normal protein, “high” Phe), liberalized Phe-restricted (33% natural protein restriction), or severe Phe-restricted (75% natural protein restriction) diet with protein substitutes for 10 weeks (n = 14 per group). Their behavioral performance was examined in an open field test, novel and spatial object location tests, and a balance beam. Fecal samples were collected and sequenced for the bacterial 16S ribosomal RNA (rRNA) region. Results indicated that PKU on a high Phe diet reduced Shannon diversity significantly and altered the microbiome composition compared with wild-type animals. Phe-restriction prevented this loss in Shannon diversity but changed community composition even more than the high-Phe diet, depending on the severity of the restriction. Moreover, on a taxonomic level, we observed the highest number of differentially abundant genera in animals that received 75% Phe-restriction. Based on correlation analyses with differentially abundant taxa, the families Entereococacceae, Erysipelotrichaceae, Porphyromonadaceae, and the genus Alloprevotella showed interesting relationships with either plasma Phe levels and/or object memory. According to our results, these bacterial taxa could be good candidates to start examining the microbial metabolic potential and probiotic properties in the context of PKU. We conclude that PKU leads to an altered gut microbiome composition in mice, which is least severe on a liberalized Phe-restricted diet. This may suggest that the current Phe-restricted diet for PKU patients could be optimized by taking dietary effects on the microbiome into account

Long-term dietary intervention with low Phe and/or a specific nutrient combination improve certain aspects of brain functioning in phenylketonuria (PKU)

Introduction In phenylketonuria (PKU), a gene mutation in the phenylalanine metabolic pathway causes accumulation of phenylalanine (Phe) in blood and brain. Although early introduction of a Phe-restricted diet can prevent severe symptoms from developing, patients who are diagnosed and treated early still experience deficits in cognitive functioning indicating shortcomings of current treatment. In the search for new and/or additional treatment strategies, a specific nutrient combination (SNC) was postulated to improve brain function in PKU. In this study, a long-term dietary intervention with a low-Phe diet, a specific combination of nutrients designed to improve brain function, or both concepts together was investigated in male and female BTBR PKU and WT mice. Material & methods 48 homozygous wild-types (WT, +/+) and 96 PKU BTBRPah2 (-/-) male and female mice received dietary interventions from postnatal day 31 till 10 months of age and were distributed in the following six groups: high Phe diet (WT C-HP, PKU C-HP), high Phe plus specific nutrient combination (WT SNC-HP, PKU SNC-HP), PKU low-Phe diet (PKU C-LP), and PKU low-Phe diet plus specific nutrient combination (PKU SNC- LP). Memory and motor function were tested at time points 3, 6, and 9 months after treatment initiation in the open field (OF), novel object recognition test (NOR), spatial object recognition test (SOR), and the balance beam (BB). At the end of the experiments, brain neurotransmitter concentrations were determined. Results In the NOR, we found that PKU mice, despite being subjected to high Phe conditions, could master the task on all three time points when supplemented with SNC. Under low Phe conditions, PKU mice on control diet could master the NOR at all three time points, while PKU mice on the SNC supplemented diet could master the task at time points 6 and 9 months. SNC supplementation did not consistently influence the performance in the OF, SOR or BB in PKU mice. The low Phe diet was able to normalize concentrations of norepinephrine and serotonin; however, these neurotransmitters were not influenced by SNC supplementation. Conclusion This study demonstrates that both a long-lasting low Phe diet, the diet enriched with SNC, as well as the combined diet was able to ameliorate some, but not all of these PKU-induced abnormalities. Specifically, this study is the first long-term intervention study in BTBR PKU mice that shows that SNC supplementation can specifically improve novel object recognition

Single amino acid supplementation in aminoacidopathies:a systematic review

Aminoacidopathies are a group of rare and diverse disorders, caused by the deficiency of an enzyme or transporter involved in amino acid metabolism. For most aminoacidopathies, dietary management is the mainstay of treatment. Such treatment includes severe natural protein restriction, combined with protein substitution with all amino acids except the amino acids prior to the metabolic block and enriched with the amino acid that has become essential by the enzymatic defect. For some aminoacidopathies, supplementation of one or two amino acids, that have not become essential by the enzymatic defect, has been suggested. This so-called single amino acid supplementation can serve different treatment objectives, but evidence is limited. The aim of the present article is to provide a systematic review on the reasons for applications of single amino acid supplementation in aminoacidopathies treated with natural protein restriction and synthetic amino acid mixtures

Blood and brain biochemistry and behaviour in NTBC and dietary treated tyrosinemia type 1 mice

Tyrosinemia type 1 (TT1) is a rare metabolic disease caused by a defect in the tyrosine degradation pathway. Neurocognitive deficiencies have been described in TT1 patients, that have, among others, been related to changes in plasma large neutral amino acids (LNAA) that could result in changes in brain LNAA and neurotransmitter concentrations. Therefore, this project aimed to investigate plasma and brain LNAA, brain neurotransmitter concentrations and behavior in C57 Bl/6 fumarylacetoacetate hydrolase deficient (FAH-/-) mice treated with 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and/or diet and wild-type mice. Plasma and brain tyrosine concentrations were clearly increased in all NTBC treated animals, even with diet (p <0.001). Plasma and brain phenylalanine concentrations tended to be lower in all FAH-/- mice. Other brain LNAA, were often slightly lower in NTBC treated FAH-/- mice. Brain neurotransmitter concentrations were usually within a normal range, although serotonin was negatively correlated with brain tyrosine concentrations (p <0.001). No clear behavioral differences between the different groups of mice could be found. To conclude, this is the first study measuring plasma and brain biochemistry in FAH-/- mice. Clear changes in plasma and brain LNAA have been shown. Further research should be done to relate the biochemical changes to neurocognitive impairments in TT1 patients

Heterogeneous clinical spectrum of DNAJC12-deficient hyperphenylalaninemia:From attention deficit to severe dystonia and intellectual disability

BACKGROUND: Autosomal recessive mutations in DNAJC12, encoding a cochaperone of HSP70 with hitherto unknown function, were recently described to lead to hyperphenylalaninemia, central monoamine neurotransmitter (dopamine and serotonin) deficiency, dystonia and intellectual disability in six subjects affected by homozygous variants. OBJECTIVE: Patients exhibiting hyperphenylalaninemia in whom deficiencies in hepatic phenylalanine hydroxylase and tetrahydrobiopterin cofactor metabolism had been excluded were subsequently analysed for DNAJC12 variants. METHODS: To analyse DNAJC12, genomic DNA from peripheral blood (Sanger sequencing), as well as quantitative messenger RNA (Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)) and protein expression (Western blot) from primary skin fibroblasts were performed. RESULTS: We describe five additional patients from three unrelated families with homozygosity/compound heterozygosity in DNAJC12 with three novel variants: c.85delC/p.Gln29Lysfs*38, c.596G>T/p.*199Leuext*42 and c.214C>T/p.(Arg72*). In contrast to previously reported DNAJC12-deficient patients, all five cases showed a very mild neurological phenotype. In two subjects, cerebrospinal fluid and primary skin fibroblasts were analysed showing similarly low 5-hydroxyindolacetic acid and homovanillic acid concentrations but more reduced expressions of mRNA and DNAJC12 compared with previously described patients. All patients responded to tetrahydrobiopterin challenge by lowering blood phenylalanine levels. CONCLUSIONS: DNAJC12 deficiency appears to result in a more heterogeneous neurological phenotype than originally described. While early identification and institution of treatment with tetrahydrobiopterin and neurotransmitter precursors is crucial to ensure optimal neurological outcome in DNAJC12-deficient patients with a severe phenotype, optimal treatment for patients with a milder phenotype remains to be defined

The behavioral consequence of phenylketonuria in mice depends on the genetic background

To unravel the role of gene mutations in the healthy and the diseased state, countless studies have tried to link genotype with phenotype. However, over the years, it became clear that the strain of mice can influence these results. Nevertheless, identical gene mutations in different strains are often still considered equals. An example of this, is the research done in phenylketonuria (PKU), an inheritable metabolic disorder. In this field, a PKU mouse model (either on a BTBR or C57Bl/6 background) is often used to examine underlying mechanisms of the disease and/or new treatment strategies. Both strains have a point mutation in the gene coding for the enzyme phenylalanine hydroxylase which causes toxic concentrations of the amino acid phenylalanine in blood and brain, as found in PKU patients. Although the mutation is identical and therefore assumed to equally affect physiology and behavior in both strains, no studies directly compared the two genetic backgrounds to test this assumption. Therefore, this study compared the BTBR and C57Bl/6 wild-type and PKU mice on PKU-relevant amino acid- and neurotransmitter levels and at a behavioral level. The behavioral paradigms were selected from previous literature on the PKU mouse model and address four domains, namely 1) activity levels, 2) motor performance, 3) anxiety and/or depression-like behavior, and 4) learning and memory. The results of this study showed comparable biochemical changes in phenylalanine and neurotransmitter concentrations. In contrast, clear differences in behavioral outcome between the strains in all four above-mentioned domains were found, most notably in the learning and memory domain. The outcome in this domain seem to be primarily due to factors inherent to the genetic background of the mouse and much less by differences in PKU-specific biochemical parameters in blood and brain. The difference in behavioral outcome between PKU of both strains emphasizes that the consequence of the PAH mutation is influenced by other factors than Phe levels alone. Therefore, future research should consider these differences when choosing one of the genetic strains to investigate the pathophysiological mechanism underlying PKU-related behavior, especially when combined with new treatment strategies

Untreated PKU patients without intellectual disability: what do they teach us?

Phenylketonuria (PKU) management is aimed at preventing neurocognitive and psychosocial dysfunction by keeping plasma phenylalanine concentrations within the recommended target range. It can be questioned, however, whether universal plasma phenylalanine target levels would result in optimal neurocognitive outcomes for all patients, as similar plasma phenylalanine concentrations do not seem to have the same consequences to the brain for each PKU individual. To better understand the inter-individual differences in brain vulnerability to high plasma phenylalanine concentrations, we aimed to identify untreated and/or late-diagnosed PKU patients with near-normal outcome, despite high plasma phenylalanine concentrations, who are still alive. In total, we identified 16 such cases. While intellectual functioning in these patients was relatively unaffected, they often did present other neurological, psychological, and behavioral problems. Thereby, these "unusual" PKU patients show that the classical symptomatology of untreated or late-treated PKU may have to be rewritten. Moreover, these cases show that a lack of intellectual dysfunction despite high plasma phenylalanine concentrations does not necessarily imply that these high phenylalanine concentrations have not been toxic to the brain. Also, these cases may suggest that different mechanisms are involved in PKU pathophysiology, of which the relative importance seems to differ between patients and possibly also with increasing age. Further research should aim to better distinguish PKU patients with respect to their cerebral effects to high plasma phenylalanine concentrations

Untreated PKU Patients without Intellectual Disability: What Do They Teach Us?

Phenylketonuria (PKU) management is aimed at preventing neurocognitive and psychosocial dysfunction by keeping plasma phenylalanine concentrations within the recommended target range. It can be questioned, however, whether universal plasma phenylalanine target levels would result in optimal neurocognitive outcomes for all patients, as similar plasma phenylalanine concentrations do not seem to have the same consequences to the brain for each PKU individual. To better understand the inter-individual differences in brain vulnerability to high plasma phenylalanine concentrations, we aimed to identify untreated and/or late-diagnosed PKU patients with near-normal outcome, despite high plasma phenylalanine concentrations, who are still alive. In total, we identified 16 such cases. While intellectual functioning in these patients was relatively unaffected, they often did present other neurological, psychological, and behavioral problems. Thereby, these "unusual" PKU patients show that the classical symptomatology of untreated or late-treated PKU may have to be rewritten. Moreover, these cases show that a lack of intellectual dysfunction despite high plasma phenylalanine concentrations does not necessarily imply that these high phenylalanine concentrations have not been toxic to the brain. Also, these cases may suggest that different mechanisms are involved in PKU pathophysiology, of which the relative importance seems to differ between patients and possibly also with increasing age. Further research should aim to better distinguish PKU patients with respect to their cerebral effects to high plasma phenylalanine concentrations

Large neutral amino acid supplementation exerts its effect through three synergistic mechanisms:Proof of principle in phenylketonuria mice

Phenylketonuria (PKU) was the first disorder in which severe neurocognitive dysfunction could be prevented by dietary treatment. However, despite this effect, neuropsychological outcome in PKU still remains suboptimal and the phenylalanine-restricted diet is very demanding. To improve neuropsychological outcome and relieve the dietary restrictions for PKU patients, supplementation of large neutral amino acids (LNAA) is suggested as alternative treatment strategy that might correct all brain biochemical disturbances caused by high blood phenylalanine, and thereby improve neurocognitive functioning.As a proof-of-principle, this study aimed to investigate all hypothesized biochemical treatment objectives of LNAA supplementation (normalizing brain phenylalanine, non-phenylalanine LNAA, and monoaminergic neurotransmitter concentrations) in PKU mice.C57Bl/6 Pah-enu2 (PKU) mice and wild-type mice received a LNAA supplemented diet, an isonitrogenic/isocaloric high-protein control diet, or normal chow. After six weeks of dietary treatment, blood and brain amino acid and monoaminergic neurotransmitter concentrations were assessed.In PKU mice, the investigated LNAA supplementation regimen significantly reduced blood and brain phenylalanine concentrations by 33% and 26%, respectively, compared to normal chow (p<0.01), while alleviating brain deficiencies of some but not all supplemented LNAA. Moreover, LNAA supplementation in PKU mice significantly increased brain serotonin and norepinephrine concentrations from 35% to 71% and from 57% to 86% of wild-type concentrations (p<0.01), respectively, but not brain dopamine concentrations (p = 0.307).This study shows that LNAA supplementation without dietary phenylalanine restriction in PKU mice improves brain biochemistry through all three hypothesized biochemical mechanisms. Thereby, these data provide proof-of-concept for LNAA supplementation as a valuable alternative dietary treatment strategy in PKU. Based on these results, LNAA treatment should be further optimized for clinical application with regard to the composition and dose of the LNAA supplement, taking into account all three working mechanisms of LNAA treatment