Tetrahydrobiopterin deficiencies: Lesson from clinical experience.

OBJECTIVES: The present study describes clinical, biochemical, molecular genetic data, current treatment strategies and follow-up in nine patients with tetrahydrobiopterin (BH4) deficiency due to various inherited genetic defects. METHODS: We analyzed clinical, biochemical, and molecular data of nine patients with suspected BH4 deficiency. All patients were diagnosed at Ege University Faculty of Medicine in Izmir, Turkey and comprised data collected from 2006 to 2019. The diagnostic laboratory examinations included blood phenylalanine and urinary or plasma pterins, dihydropteridine reductase (DHPR) enzyme activity measurement in dried blood spots, folic acid and monoamine neurotransmitter metabolites in cerebrospinal fluid, as well as DNA sequencing. RESULTS: Among the nine patients, we identified one with autosomal recessive GTP cyclohydrolase I (ar GTPCH) deficiency, two with 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency, three with sepiapterin reductase (SR) deficiency, and three with DHPR deficiency. Similar to previous observations, the most common clinical symptoms are developmental delay, intellectual disability, and movement disorders. All patients received treatment with l-dopa and 5-hydroxytryptophan, while only the ar GTPCH, the PTPS, and one DHPR deficient patients were supplemented in addition with BH4. The recommended dose range varies among patients and depends on the type of disease. The consequences of BH4 deficiencies are quite variable; however, early diagnosis and treatment will improve outcomes. CONCLUSIONS: As BH4 deficiencies are rare group of treatable neurometabolic disorders, it is essential to diagnose the underlying (genetic) defect in newborns with hyperphenylalaninemia. Irreversible brain damage and progressive neurological deterioration may occur in untreated or late diagnosed patients. Prognosis could be satisfying in the cases with early diagnose and treatment.

Oxidative Stress, Folate Receptor Autoimmunity, and CSF Findings in Severe Infantile Autism.

BACKGROUND: Biomarkers such as oxidative stress, folate receptor alpha (FRα) autoimmunity, and abnormal brain serotonin turnover are common in autism. METHODS: Oxidative stress biomarkers with pro- and antioxidants were measured in the severe form of infantile autism (n = 38) and controls (n = 24). Children and parents had repeated testing for serum FR autoantibodies, spinal fluid dopamine and serotonin metabolites, pterins, and N5-methyltetrahydrofolate (MTHF). Statistical analysis assessed correlations between variables. Genetic analysis included the SLC6A4 and SLC29A4 genes encoding synaptic serotonin reuptake proteins. RESULTS: Compared to controls, the autism group showed a significant increase in oxidative DNA damage in lymphocytes, plasma ceruloplasmin and copper levels with a high copper/zinc ratio, thiol proteins, and superoxide dismutase (SOD) activity. Vitamin C levels were significantly diminished. In most autistic patients, the vitamin A (64%) and D (70%) levels were low. Serum FR autoantibodies fluctuating over 5-7 week periods presented in 68% of all autistic children, 41% of parents vs. 3.3% of control children and their parents. CSF showed lowered serotonin 5-hydroxyindole acetic acid (5HIAA) metabolites in 13 (34%), a low 5HIAA to HVA (dopamine metabolite) ratio in 5 (13%), low 5HIAA and MTHF in 2 (5%), and low MTHF in 8 patients (21%). A known SLC6A4 mutation was identified only in 1 autistic child with low CSF 5HIAA and a novel SLC29A4 mutation was identified in identical twins. Low CSF MTHF levels among only 26% of subjects can be explained by the fluctuating FR antibody titers. Two or more aberrant pro-oxidant and/or antioxidant factors predisposed to low CSF serotonin metabolites. Three autistic children having low CSF 5HIAA and elevated oxidative stress received antioxidative supplements followed by CSF 5HIAA normalisation. CONCLUSION: In autism, we found diverse combinations for FR autoimmunity and/or oxidative stress, both amenable to treatment. Parental and postnatal FR autoantibodies tend to block folate passage to the brain affecting folate-dependent pathways restored by folinic acid treatment, while an abnormal redox status tends to induce reduced serotonin turnover, corrected by antioxidant therapy. Trial Registration. The case-controlled study was approved in 2008 by the IRB at Liège University (Belgian Number: B70720083916). Lay Summary. Children with severe infantile autism frequently have serum folate receptor autoantibodies that block the transport of the essential vitamin folate across the blood-brain barrier to the brain. Parents are often asymptomatic carriers of these serum folate receptor autoantibodies, which in mothers can block folate passage across the placenta to their unborn child. This folate deficiency during the child's intrauterine development may predispose to neural tube defects and autism. Oxidative stress represents a condition with the presence of elevated toxic oxygen derivatives attributed to an imbalance between the formation and protection against these toxic reactive oxygen derivatives. Oxidative stress was found to be present in autistic children where these reactive oxygen derivatives can cause damage to DNA, which changes DNA function and regulation of gene expression. In addition, excessive amounts of these toxic oxygen derivatives are likely to damage the enzyme producing the neuromessenger serotonin in the brain, diminished in about 1/3 of the autistic children. Testing children with autism for oxidative stress and its origin, as well as testing for serum folate receptor autoantibodies, could open new approaches towards more effective treatments.

Tyrosine hydroxylase deficiency-Clinical insights and a novel deletion in TH gene in an Indian patient.

Tyrosine hydroxylase deficiency is a rare autosomal recessive, treatable disorder of neurotransmission. Fewer than 100 cases have been reported so far. We present a case of a 10-month-old infant who was symptomatic since 5 months of age and who received an initial diagnosis of infantile tremor syndrome. She presented with rest tremor, decreased facial expression, global hypokinesia, and later on with oculogyric crisis and dystonia. This diagnosis was revised after confirmation of tyrosine hydroxylase deficiency by CSF neurotransmitter analysis. Genetic studies revealed one previously reported missense variant, p.Thr399Met, and another large deletion starting upstream of exon 1 and encompassing exon 1. She was started on treatment with escalating doses of L-Dopa/Carbidopa, with folinic acid supplementation. At 3.5 years of age, her cognitive functioning and development is appropriate for age. There is complete subsidence of dystonia and oculogyric episodes. She has occasional chorieform movements which appear to be drug related.

Treatment of Cystathionine ß-Synthase Deficiency in Mice Using a Minicircle-Based Naked DNA Vector.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by extremely elevated total homocysteine (tHcy) in the blood. Patients diagnosed with CBS deficiency have a variety of clinical problems, including dislocated lenses, osteoporosis, cognitive and behavioral issues, and a significantly increased risk of thrombosis. Current treatment strategies involve a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine. Here, a mouse model for CBS deficiency (Tg-I278T Cbs-/-) was used to evaluate the potential of minicircle-based naked DNA gene therapy to treat CBS deficiency. A 2.3 kb DNA-minicircle containing the liver-specific P3 promoter driving the human CBS cDNA (MC.P3-hCBS) was delivered into Tg-I278T Cbs-/- mice via a single hydrodynamic tail vein injection. Mean serum tHcy decreased from 351 µM before injection to 176 µM 7 days after injection (p = 0.0005), and remained decreased for at least 42 days. Western blot analysis reveals significant minicircle-directed CBS expression in the liver tissue. Liver CBS activity increased 34-fold (12.8 vs. 432 units; p = 0.0004) in MC.P3-hCBS-injected animals. Injection of MC.P3-hCBS in young mice, subsequently followed for 202 days, showed that the vector can ameliorate the mouse homocystinuria alopecia phenotype. The present findings show that minicircle-based gene therapy can lower tHcy in a mouse model of CBS deficiency.

Tetrahydrobiopterin treatment reduces brain L-Phe but only partially improves serotonin in hyperphenylalaninemic ENU1/2 mice.

Hyperphenylalaninemia (HPA) caused by hepatic phenylalanine hydroxylase (PAH) deficiency has severe consequences on brain monoamine neurotransmitter metabolism. We have studied monoamine neurotransmitter status and the effect of tetrahydrobiopterin (BH4) treatment in Pahenu1/enu2 (ENU1/2) mice, a model of partial PAH deficiency. These mice exhibit elevated blood L-phenylalanine (L-Phe) concentrations similar to that of mild hyperphenylalaninemia (HPA), but brain levels of L-Phe are still ~5-fold elevated compared to wild-type. We found that brain L-tyrosine, L-tryptophan, BH4 cofactor and catecholamine concentrations, and brain tyrosine hydroxylase (TH) activity were normal in these mice but that brain serotonin, 5-hydroxyindolacetic acid (5HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) content, and brain TH protein, as well as tryptophan hydroxylase type 2 (TPH2) protein levels and activity were reduced in comparison to wild-type mice. Parenteral L-Phe loading conditions did not lead to significant changes in brain neurometabolite concentrations. Remarkably, enteral BH4 treatment, which normalized brain L-Phe levels in ENU1/2 mice, lead to only partial recovery of brain serotonin and 5HIAA concentrations. Furthermore, indirect evidence indicated that the GTP cyclohydrolase I (GTPCH) feedback regulatory protein (GFRP) complex may be a sensor for brain L-Phe elevation to ameliorate the toxic effects of HPA. We conclude that BH4 treatment of HPA toward systemic L-Phe lowering reverses elevated brain L-Phe content but the recovery of TPH2 protein and activity as well as serotonin levels is suboptimal, indicating that patients with mild HPA and mood problems (depression or anxiety) treated with the current diet may benefit from supplementation with BH4 and 5-OH-tryptophan.

DNAJC12 deficiency: A new strategy in the diagnosis of hyperphenylalaninemias.

Patients with hyperphenylalaninemia (HPA) are detected through newborn screening for phenylketonuria (PKU). HPA is known to be caused by deficiencies of the enzyme phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH4). Current guidelines for the differential diagnosis of HPA would, however, miss a recently described DNAJC12 deficiency. The co-chaperone DNAJC12 is, together with the 70kDa heat shock protein (HSP70), responsible for the proper folding of PAH. All DNAJC12-deficient patients investigated to date responded to a challenge with BH4 by lowering their blood phenylalanine levels. In addition, the patients presented with low levels of biogenic amine in CSF and responded to supplementation with BH4, L-dopa/carbidopa and 5-hydroxytryptophan. The phenotypic spectrum ranged from mild autistic features or hyperactivity to severe intellectual disability, dystonia and parkinsonism. Late diagnosis result in permanent neurological disability, while early diagnosed and treated patients develop normally. Molecular diagnostics for DNAJC12 variants are thus mandatory in all patients in which deficiencies of PAH and BH4 are genetically excluded.

Neurological improvement following intravenous high-dose folinic acid for cerebral folate transporter deficiency caused by FOLR-1 mutation.

BACKGROUND: Cerebral folate transporter deficiency caused by FOLR-1 mutations has been described in 2009. This condition is characterized by a 5MTHF level <5 nmol/l in the CSF, along with regression of acquisition in the second year of life, ataxia, and refractory myoclonic epilepsy. Oral or intravenous folinic acid (5-formyltetrahydrofolate) treatment has been shown to improve clinical status. CASE PRESENTATION: We present the cases of two sisters with cerebral folate transport deficiency caused by mutation in the folate receptor 1 (FOLR1) gene (MIM *136430). Following recommendations, we administered oral folinic acid at 5 mg/kg/day, resulting in some initial clinical improvement, yet severe epilepsy persisted. During treatment, cerebrospinal fluid (CSF) analysis revealed normal 5-methyltetrahydrofolate (5MTHF) levels (60.1 nmol/l; normal range: 53-182 nmol/l). Epilepsy proved difficult to control and the younger patient exhibited neurological regression. We then administered high-dose folinic acid intravenously over 3 days (6 mg/kg/day for 24 h, then 12 mg/kg/day for 48 h), which significantly improved clinical status and epilepsy. CSF analysis revealed high 5MTHF levels following intravenous infusion (180 nmol/l). Treatment continued with monthly intravenous administrations of 20-25 mg/kg folinic acid. At 2 years post-treatment, clinical improvement was confirmed. CONCLUSIONS: This report illustrates that cerebral folate transporter deficiency caused by FOLR-1 mutations is a treatable condition and can potentially be cured by folinic acid treatment. As already reported, early effective treatment is known to improve outcomes in affected children. In our study, intravenous high-dose folinic acid infusions appeared to optimize clinical response.

Mildly compromised tetrahydrobiopterin cofactor biosynthesis due to Pts variants leads to unusual body fat distribution and abdominal obesity in mice.

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.

High dose sapropterin dihydrochloride therapy improves monoamine neurotransmitter turnover in murine phenylketonuria (PKU).

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters, dopamine and serotonin, have been implicated in the pathophysiology of neuropsychiatric dysfunction in phenylketonuria (PKU). Increased brain phenylalanine concentration likely competitively inhibits the activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH), the rate limiting steps in dopamine and serotonin synthesis respectively. Tetrahydrobiopterin (BH4) is a required cofactor for TH and TPH activity. Our hypothesis was that treatment of hyperphenylalaninemic Pah(enu2/enu2) mice, a model of human PKU, with sapropterin dihydrochloride, a synthetic form of BH4, would stimulate TH and TPH activities leading to improved dopamine and serotonin synthesis despite persistently elevated brain phenylalanine. Sapropterin (20, 40, or 100mg/kg body weight in 1% ascorbic acid) was administered daily for 4 days by oral gavage to Pah(enu2/enu2) mice followed by measurement of brain biopterin, phenylalanine, tyrosine, tryptophan and monoamine neurotransmitter content. A significant increase in brain biopterin content was detected only in mice that had received the highest sapropterin dose, 100mg/kg. Blood and brain phenylalanine concentrations were unchanged by sapropterin therapy. Sapropterin therapy also did not alter the absolute amounts of dopamine and serotonin in brain but was associated with increased homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), dopamine and serotonin metabolites respectively, in both wild type and Pah(enu2/enu2) mice. Oral sapropterin therapy likely does not directly affect central nervous system monoamine synthesis in either wild type or hyperphenylalaninemic mice but may stimulate synaptic neurotransmitter release and subsequent metabolism.

Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver.

UNLABELLED: Host immune response to viral vectors, persistence of nonintegrating vectors, and sustained transgene expression are among the major challenges in gene therapy. To overcome these hurdles, we successfully used minicircle (MC) naked-DNA vectors devoid of any viral or bacterial sequences for the long-term treatment of murine phenylketonuria, a model for a genetic liver defect. MC-DNA vectors expressed the murine phenylalanine hydroxylase (Pah) complementary DNA (cDNA) from a liver-specific promoter coupled to a de novo designed hepatocyte-specific regulatory element, designated P3, which is a cluster of evolutionary conserved transcription factor binding sites. MC-DNA vectors were subsequently delivered to the liver by a single hydrodynamic tail vein (HTV) injection. The MC-DNA vector normalized blood phenylalanine concomitant with reversion of hypopigmentation in a dose-dependent manner for more than 1 year, whereas the corresponding parental plasmid did not result in any phenylalanine clearance. MC vectors persisted in an episomal state in the liver consistent with sustained transgene expression and hepatic PAH enzyme activity without any apparent adverse effects. Moreover, 14-20% of all hepatocytes expressed transgenic PAH, and the expression was observed exclusively in the liver and predominately around pericentral areas of the hepatic lobule, while there was no transgene expression in periportal areas. CONCLUSION: This study demonstrates that MC technology offers an improved safety profile and has the potential for the genetic treatment of liver diseases.

The mechanism of BH4 -responsive hyperphenylalaninemia--as it occurs in the ENU1/2 genetic mouse model.

The Pah(enu1/enu2) (ENU1/2) mouse is a heteroallelic orthologous model displaying blood phenylalanine (Phe) concentrations characteristic of mild hyperphenylalaninemia. ENU1/2 mice also have reduced liver phenylalanine hydroxylase (PAH) protein content (∼20% normal) and activity (∼2.5% normal). The mutant PAH protein is highly ubiquitinated, which is likely associated with its increased misfolding and instability. The administration of a single subcutaneous injection of l-Phe (1.1 mg l-Phe/g body weight) leads to an approximately twofold to threefold increase of blood Phe and phenylalanine/tyrosine (Phe/Tyr) ratio, and a 1.6-fold increase of both nonubiquitinated PAH protein content and PAH activity. It also results in elevated concentrations of liver 6R-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), potentially through the influence of Phe on GTP cyclohydrolase I and its feedback regulatory protein. The increased BH(4) content seems to stabilize PAH. Supplementing ENU1/2 mice with BH(4) (50 mg/kg/day for 10 days) reduces the blood Phe/Tyr ratio within the mild hyperphenylalaninemic range; however, PAH content and activity were not elevated. It therefore appears that BH(4) supplementation of ENU1/2 mice increases Phe hydroxylation levels through a kinetic rather than a chaperone stabilizing effect. By boosting blood Phe concentrations, and by BH(4) supplementation, we have revealed novel insights into the processing and regulation of the ENU1/2-mutant PAH.

Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l-DOPA responsive and non-responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription-translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH(4) supplementation to mice increased TH activity and protein levels in brain extracts, while the Th-mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding-aid effect of BH(4) and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH(4) crosses the blood-brain barrier and therapeutic regimes based on BH(4) supplementation should thus consider the effect on TH. Furthermore, BH(4) supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.