Early-onset autosomal dominant GTP-cyclohydrolase I deficiency: Diagnostic delay and residual motor signs.

OBJECTIVE: Autosomal dominant (AD) guanosine triphosphate cyclohydrolase 1 (GCH1) deficiency is the most common cause of dopa-responsive dystonia (DRD). Patients with GCH1 deficiency are likely to experience diagnostic delay, but its consequences have not been described thoroughly in patients with early-onset disease. We describe the diagnostic delay and residual motor signs (RMS) observed in patients with early-onset (before 15 years of age) disease. METHODS: Twelve patients with early-onset AD GCH1 deficiency from a single center were included in the case series analysis. For the meta-analysis, the PubMed database was searched for articles on early-onset AD GCH1 deficiency published from 1995 to 2019. RESULTS: In the case series, the mean duration of diagnostic delay was 5.6 years. Two patients exhibited RMS, and four patients underwent orthopedic surgery. The literature search yielded 137 AD GCH1 deficiency cases for review; gait disturbance was reported in 92.7% of patients, diurnal fluctuation of symptoms in 91.9%, and RMS in 39%. The mean duration of diagnostic delay was 14.6 years overall: 12.0 years in RMS-negative patients and 21.2 years in RMS-positive patients. CONCLUSIONS: Diagnostic delay in early-onset AD GCH1 deficiency is more closely associated with later RMS. Early clinical suspicion, timely diagnosis, and levodopa treatment may reduce the occurrence of RMS in patients with early-onset AD GCH1 deficiency.

GTP-Cyclohydrolase I deficiency presenting as malignant hyperphenylalaninemia, recurrent hyperthermia and progressive neurological dysfunction in a South Asian child - a case report.

BACKGROUND: Tetrahydrobiopterin (BH4) deficiencies are disorders affecting phenylalanine homeostasis, and catecholamine and serotonin biosynthesis. GTP-Cyclohydrolase I deficiency (OMIM 600225) is an extremely rare variant of inborn error of BH4 synthesis which exists in recessive and dominant forms. The recessive form presents with complex neurological and autonomic dysfunction whilst the dominant form presents as Dopa-responsive dystonia. CASE PRESENTATION: We describe a South Asian child who initially presented with neurological dysfunction and recurrent vomiting and later developed recurrent hyperthermia for several months. The child did not have screening for hyperphenylalaninemia at birth and was found to have marked hyperphenylalaninemia after clinical presentation at 5 months. Further evaluation revealed BH4 deficiency. GTP-Cyclohydrolase I deficiency (GTPCH) was identified based on normal dihydro pteridine reductase activity and markedly reduced neopterin in dried blood spot test. After institution of treatment and control of high phenylalanine levels, clinical deterioration decelerated yet with noticeable residual neurological dysfunction. CONCLUSION: To authors' knowledge, this is first report of GTPCH deficiency in a South Asian child. The case highlights practical issues regarding diagnosis of GTPCH deficiency, especially in countries without broader universal newborn screening programs for early detection of inherited metabolic disorders. Testing for GTPCH deficiency should be considered for patients with unexplained neurological and autonomic symptoms following initial metabolic screen.

Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation.

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics "biotin-switch" approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis.

Roles for endothelial cell and macrophage Gch1 and tetrahydrobiopterin in atherosclerosis progression.

Aims: GTP cyclohydrolase I catalyses the first and rate-limiting reaction in the synthesis of tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthases (NOS). Both eNOS and iNOS have been implicated in the progression of atherosclerosis, with opposing effects in eNOS and iNOS knockout mice. However, the pathophysiologic requirement for BH4 in regulating both eNOS and iNOS function, and the effects of loss of BH4 on the progression of atherosclerosis remains unknown. Methods and results: Hyperlipidemic mice deficient in Gch1 in endothelial cells and leucocytes were generated by crossing Gch1fl/flTie2cre mice with ApoE-/- mice. Deficiency of Gch1 and BH4 in endothelial cells and myeloid cells was associated with mildly increased blood pressure. High fat feeding for 6 weeks in Gch1fl/flTie2CreApoE-/- mice resulted in significantly decreased circulating BH4 levels, increased atherosclerosis burden and increased plaque macrophage content. Gch1fl/flTie2CreApoE-/- mice showed hallmarks of endothelial cell dysfunction, with increased aortic VCAM-1 expression and decreased endothelial cell dependent vasodilation. Furthermore, loss of BH4 from pro-inflammatory macrophages resulted in increased foam cell formation and altered cellular redox signalling, with decreased expression of antioxidant genes and increased reactive oxygen species. Bone marrow chimeras revealed that loss of Gch1 in both endothelial cells and leucocytes is required to accelerate atherosclerosis. Conclusion: Both endothelial cell and macrophage BH4 play important roles in the regulation of NOS function and cellular redox signalling in atherosclerosis.

Disorders of biopterin metabolism.

Defects in the metabolism or regeneration of tetrahydrobiopterin (BH4) were initially discovered in patients with hyperphenylalaninaemia who had progressive neurological deterioration despite optimal metabolic control (malignant hyperphenylalaninaemia). BH4 is an essential cofactor not only for phenylalanine hydroxylase, but also for tyrosine and two tryptophan hydroxylases, three nitric oxide synthases, and glyceryl-ether monooxygenase. Defective activity of tyrosine and tryptophan hydroxylases explains the neurological deterioration in patients with BH4 deficiency with progressive mental and physical retardation, central hypotonia and peripheral spasticity, seizures and microcephaly. Five separate genetic conditions affect BH4 synthesis or regeneration: deficiency of GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase, sepiapterin reductase, dihydropteridine reductase (DHPR) and pterin-4alpha-carbinolamine dehydratase. Only the latter of these conditions is relatively benign and is associated with transient hyperphenylalaninaemia. All these conditions can be identified in newborns by an elevated phenylalanine, with the exception of sepiapterin reductase and the dominant form of GTP cyclohydrolase I deficiency that results in biopterin deficiency/insufficiency only in the brain. Diagnosis relies on the measurement of pterin metabolites in urine, dihydropteridine reductase in blood spots, neurotransmitters and pterins in the CSF and on the demonstration of reduced enzyme activity (red blood cells or fibroblasts) or causative mutations in the relative genes. The outcome of BH4 deficiency is no longer malignant if therapy is promptly initiated to reduce plasma phenylalanine levels and replace missing neurotransmitters. This is accomplished by a special diet and/or BH4 supplements and administration of L-dopa, carbidopa, 5-hydroxytryptophan, and, in certain cases, a MAO-B inhibitor. Patients with DHPR deficiency also require folinic acid supplements, since DHPR may help in maintaining folate in the tetrahydro form. Several patients with BH4 deficiency treated since the newborn period have reached adult age with good outcome.

Tetrahydrobiopterin availability, nitric oxide metabolism and glutathione status in the hph-1 mouse; implications for the pathogenesis and treatment of tetrahydrobiopterin deficiency states.

Tetrahydrobiopterin (BH4) is an essential cofactor for all isoforms of nitric oxide synthase. While it is well established that BH4 deficiency states are associated with impairment of dopamine, serotonin and phenylalanine metabolism, less is known with regard to the effects of deficiency of the cofactor upon nitric oxide (NO) metabolism. In this study, we have evaluated the effects of partial BH4 deficiency upon (a) tissue availability of the antioxidant glutathione, (b) basal NO production and (c) NO generation following exposure to lipopolysaccharide (LPS), which is known to increase expression of the inducible form of nitric oxide synthase. Using the hph-1 mouse, which displays a partial BH4 deficiency owing to impaired activity of GTP cyclohydrolase, we report decreased levels of glutathione in brain and kidney and evidence for decreased basal generation of nitric oxide in the periphery (as judged by the plasma nitrate plus nitrite concentration). Following LPS administration, peripheral NO generation increases. However, the concentration of plasma nitrate plus nitrite achieved was significantly decreased in the hph-1 mouse. Furthermore, LPS administration caused loss of glutathione in both wild-type and hph-1 liver and kidney. It is concluded that cofactor replacement, sufficient to fully correct a cellular BH4 deficiency, may be of benefit to patients with inborn errors of BH4 metabolism.

Characterization of wild-type and mutants of recombinant human GTP cyclohydrolase I: relationship to etiology of dopa-responsive dystonia.

To explore the molecular etiology of two disorders caused by a defect in GTP cyclohydrolase I--hereditary progressive dystonia with marked diurnal fluctuation (HPD), also known as dopa-responsive dystonia (DRD), and autosomal recessive GTP cyclohydrolase I deficiency--we purified and analyzed recombinant human wild-type and mutant GTP cyclohydrolase I proteins expressed in Escherichia coli. Mutant proteins showed very low enzyme activities, and some mutants were eluted at a delayed volume on gel filtration compared with the recombinant wild-type. Next, we examined the GTP cyclohydrolase I protein amount by western blot analysis in phytohemagglutinin-stimulated mononuclear blood cells from HPD/DRD patients. We found a great reduction in the amount of the enzyme protein not only in one patient who had a frameshift mutation, but also in an HPD/DRD patient who had a missense mutation. These results suggest that a dominant-negative effect of chimeric protein composed of wild-type and mutant subunits is unlikely as a cause of the reduced enzyme activity in HPD/DRD patients. We suggest that reduction of the amount of the enzyme protein, which is independent of the mutation type, could be a reason for the dominant inheritance in HPD/DRD.

A missense mutation in a patient with guanosine triphosphate cyclohydrolase I deficiency missed in the newborn screening program.

A patient with guanosine triphosphate cyclohydrolase I deficiency passed the newborn phenylketonuria screening program. The characteristic clinical phenotype developed in a 5-month-old patient; elevated plasma phenylalanine, undetectable urinary pterins, and absence of the enzyme activity in a liver biopsy were present. A point mutation that results in an amino acid substitution from methionine to isoleucine at position 211 was proposed to be the cause for this new phenotypic expression of guanosine triphosphate cyclohydrolase I deficiency.

Guanosine triphosphate cyclohydrolase I deficiency: early diagnosis by routine urine pteridine screening.

A deficiency of hepatic guanosine triphosphate cyclohydrolase I is reported in a 4-month-old infant in whom positive results on a Guthrie phenylketonuria test in the neonatal period were found. Because of the significantly elevated serum phenylalanine levels a diagnosis of classical phenylketonuria was made, and dietary therapy was started. Urinary pteridine screening for cofactor variants, however, revealed extremely low levels of both neopterin and biopterin. This suggested the possibility of guanosine triphosphate cyclohydrolase I deficiency and led to additional confirmatory assays. Repeat urine, serum, and CSF pteridine profiles, combined with tetrahydrobiopterin-loading studies and the assay of guanosine triphosphate cyclohydrolase I activity in a liver biopsy, confirmed the defect. It is significant to note that the diagnosis was made before the onset of major clinical symptoms. This case illustrates the need for routine cofactor variant screening of all infants in whom hyperphenylalaninemia is diagnosed in the neonatal period.

Differential diagnosis of tetrahydrobiopterin deficiency.

Six hundred and seventy-three children (483 newborns and 190 older selected children) were screened for tetrahydrobiopterin (BH4) deficiency by HPLC of urine pterins and BH4 load test. One patient with GTP cyclohydrolase I deficiency, 36 patients with dihydrobiopterin synthetase (DHBS) deficiency (of which six were in the newborn and 30 in the older children) and 14 with dihydropteridine reductase deficiency (DHPR) were found. All 37 patients with defective BH4 biosynthesis responded to a BH4 load by lowering of the elevated serum phenylalanine concentration but four of 14 patients with DHPR deficiency did not. Measurement of DHPR activity in blood spots on Guthrie cards is recommended. Since subvariants of patients with BH4 deficiency exist, homovanillic acid, 5-hydroxyindole acetic acid, pterins, phenylalanine, and tyrosine in cerebrospinal fluid should be measured for diagnosis and the control of therapy. The activity of the phosphate-eliminating enzyme (a key enzyme in BH4 biosynthesis and part of "DHBS") was measured in human liver and activities of approx. 1 n U (mg protein)-1 were found. In the liver biopsy of a patient with DHBS deficiency no activity (less than 3% of controls) was demonstrated.