Striatal biopterin and tyrosine hydroxylase protein reduction in dopa-responsive dystonia.

OBJECTIVE: To determine the mechanism leading to striatal dopamine (DA) loss in dopa-responsive dystonia (DRD). BACKGROUND: Although mutations in the gene GCH1, coding for the tetrahydrobiopterin (BH4) biosynthetic enzyme guanosine triphosphate-cyclohydrolase I, have been identified in some patients with DRD, the actual status of brain BH4 (the cofactor for tyrosine hydroxylase [TH]) is unknown. METHODS: The authors sequenced GCH1 and measured levels of total biopterin (BP) and total neopterin (NP), TH, and dopa decarboxylase (DDC) proteins, and the DA and vesicular monoamine transporters (DAT, VMAT2) in autopsied brain of two patients with typical DRD. RESULTS: Patient 1 had two GCH1 mutations but Patient 2 had no mutation in the coding region of this gene. Striatal BP levels were markedly reduced (<20% of control subjects) in both patients and were also low in two conditions characterized by degeneration of nigrostriatal DA neurons (PD and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treated primate), whereas brain NP concentrations were selectively decreased (<45%) in the DRD patients. In the putamen, both DRD patients had severely reduced (<3%) TH protein levels but had normal concentrations of DDC protein, DAT, and VMAT2. CONCLUSIONS: The data suggest that 1) brain BH4 is decreased substantially in dopa-responsive dystonia, 2) dopa-responsive dystonia can be distinguished from degenerative nigrostriatal dopamine deficiency disorders by the presence of reduced brain neopterin, and 3) the striatal dopamine reduction in dopa-responsive dystonia is caused by decreased TH activity due to low cofactor concentration and to actual loss of TH protein. This reduction of TH protein, which might be explained by reduced enzyme stability/expression consequent to congenital BH4 deficiency, can be expected to limit the efficacy of acute BH4 administration on dopamine biosynthesis in dopa-responsive dystonia.     

Dopa-responsive dystonia: Pathological and biochemical observations in a case

We report the first neuropathological and neurochemical study of a patient with dopa-responsive dystonia. She had onset of foot dystonia at age 5 years and by age 8 years it was generalized with prominent right leg and arm involvement. On levodopa 750 mg daily she had complete symptomatic improvement that was sustained for 11 years until death. Pathological studies revealed normal numbers of hypopigmented substantia nigra neurons, normal tyrosine hydroxylase (TH) immunoreactivity and TH protein in the SN, no inclusion bodies or gliosis, and no evidence of a degenerative process in the striatum. Biochemical studies revealed reduced dopamine in the substantia nigra and striatum (8% in the putamen and 18% of control in the caudate) with a similar but not identical subregional distribution as in idiopathic Parkinson's disease. In the striatum, TH protein and TH activity was reduced, with the loss more pronounced in the putamen than the caudate. The GBR 12935 binding to DA transporter was normal in the caudate and at the lower end of the range of control values in the putamen. We conclude that disturbed dopamine synthetic capacity or a reduced arborization of striatal dopamine terminals may be the major disturbance in dopa-responsive dystonia.     

Dopa-responsive dystonia: clinical, genetic, and biochemical studies]

Dopa-responsive dystonia (DRD) is a clinical syndrome characterized by childhood-onset dystonia and a dramatic and sustained response to low doses of levodopa. There are at least three causative genes for DRD: (1) the GCH1 gene on chromosome 14q22.1-q22.2, which encodes GTP cyclohydrolase I (GTPCH), the first enzyme in the biosynthetic pathway for tetrahydrobiopterin (BH4; the essential cofactor for tyrosine hydroxylase [THI]), (2) the TH gene on 11 p15.5, coding for the enzyme TH that catalyzes the rate-limiting step in the catecholamine biosynthesis, and (3) an as yet undefined gene on 14q13 (DYT14). In reports on DRD, in which conventional genomic DNA sequencing of GCH1 was conducted in a relatively large number of pedigrees, mutations in the coding region (including the splice sites) of this gene were found in approximately 60% (range: 49-79%) of DRD families. In our series, after conducting additional GCH1 testing (Southern blotting, cDNA sequencing, etc.) and TH analysis, 86% of families with DRD or dystonia with motor delay (an intermediate phenotype between GTPCH-deficient DRD [mild] and GTPCH-deficient hyperphenylalaninemia [severe]) had identifiable GCH1 or (rarely) TH mutations. Up to the present, only one pedigree with autosomal dominant DRD linked to the DYT14 locus has been reported. Neuropathological findings (no Lewy bodies and a normal population of cells with reduced melanin in the substantia nigra) in DRD patients with GTPCH dysfunction were similar to those in a patient with DYT14 dystonia. There have been no reports of autopsied patients with TH-deficient DRD. Neurochemical data suggest that striatal dopamine reduction in GTPCH-deficient DRD is caused not only by decreased TH activity resulting from a low cofactor (BH4) level but also by actual loss of TH protein without nerve terminal loss. This TH protein reduction in the striatum, especially in the putamen, may be due to a diminished regulatory effect of BH4 on stability (rather than expression) of TH molecules or to a dysfunction of TH protein transport from the substantia nigra to the striatum. The extent of striatal TH protein loss may be critical in determining DRD symptomatology and could contribute to gender-related incomplete penetrance of GCH1 mutations in GTPCH-deficient DRD families. Notwithstanding the discovery of the three causative loci for DRD, a therapeutic trial with low doses of levodopa is still the most practical approach to the diagnosis of this treatable disorder. The trial should be considered in all children with dystonic and/or parkinsonian symptoms or with unexplained gait disorders. Analyses of total biopterin and neopterin as well as neurotransmitter metabolites in CSF appear to be useful for the diagnosis of GTPCH-deficient DRD (the major form of DRD) and of TH-deficient DRD (the mild form of TH deficiency). Findings of the precise mechanism of striatal TH protein loss in GTPCH-deficient DRD, the actual status of dopaminergic systems in TH-deficient DRD, and the novel causative gene on the DYT14 locus will better define the pathogenesis of DRD.     

Dopa-responsive dystonia]

Dopa-responsive dystonia (DRD) is a clinical syndrome characterized by childhood-onset dystonia and a dramatic and sustained response to relatively low doses of levodopa. There are at least three causative genes for DRD: 1) the GCH1 gene on chromosome 14q22.1-q22.2, coding for the enzyme GTP cyclohydrolase I (GTPCH) that catalyzes the rate-limiting step in the tetrahydrobiopterin (BH4; the cofactor for tyrosine hydroxylase [TH]) biosynthesis, 2) the TH gene on 11p15.5, and 3) an as yet undefined gene on 14q13 (DYT14). In our series, 86% of families with DRD or dystonia with motor delay (an intermediate phenotype between GTPCH-deficient DRD [mild] and GTPCH-deficient hyperphenylalaninemia [severe]) had identifiable GCH1 or (rarely) TH mutations. Neurochemical data suggest that striatal dopamine reduction in GTPCH-deficient DRD (the major form of DRD) is caused not only by decreased TH activity resulting from a low cofactor level but also by actual loss of TH protein without nerve terminal loss. This TH protein reduction in the striatum (especially in the putamen) may be due to a diminished regulatory effect of BH4 on stability of TH molecules or to a dysfunction of TH protein transport from the substantia nigra to the striatum.     

Low frequency of GCH1 and TH mutations in Parkinson's disease

BackgroundThe causes of Parkinson's disease (PD) are unknown in the majority of patients. The GCH1 gene encodes GTP-cyclohydrolase I, an important enzyme in dopamine synthesis. Co-occurrence of dopa-responsive dystonia (DRD) and a PD phenotype has been reported in families with GCH1 mutations. Recently, rare coding variants in GCH1 were found to be enriched in PD patients, indicating a role for the enzyme in the neurodegenerative process.MethodsTo further elucidate the contribution of GCH1 mutations to sporadic PD, we examined its coding exons in a targeted deep sequencing study of 509 PD patients (mean age at onset 56.7 ± 12.0 years) and 230 controls. We further included the tyrosine hydroxylase gene TH, also known to cause DRD. Gene dose assessments were performed to screen for large copy number variants in a subset of 48 patients with early-onset PD.ResultsNo putatively pathogenic GCH1 mutations were found. The frequency of rare heterozygous variants in the TH gene was 0.69% (7/1018) in the patient group and 0.22% (1/460) in the control group (p = 0.45).ConclusionsPrevious studies have found that coding variants in the GCH1 gene may be considered a risk factor for PD. Our study indicates that mutations in GCH1 are rare in late-onset PD. Several patients carried heterozygous variants in the TH gene that may affect protein function. Our study was not designed to determine with certainty if any of these variants play a role as risk factors for late-onset PD.     

GCH1 mutation in a patient with adult-onset oromandibular dystonia

The authors report a mutation in exon 5 of GCH1 in a patient with adult-onset oromandibular dystonia and no obvious family history of dystonia. The patient responded positively to treatment with L-dopa. These findings demonstrate that GCH1 mutations must be considered even in patients with dystonic symptoms not typical of dopa-responsive dystonia.     

Dopa-responsive dystonia: [18F]dopa positron emission tomography.

The syndrome of dopa-responsive dystonia comprises a minority of patients with dystonia, yet it is of considerable diagnostic importance because patients respond dramatically to L-dopa therapy. Benefits from this treatment are lasting, and the problems associated with long-term L-dopa therapy in patients with Parkinson's disease are generally absent. It has been suggested that this condition is due to a defect in the dopamine synthetic pathway, which is bypassed when patients are treated with L-dopa. We have studied [18F]dopa uptake in 6 patients with classic dopa-responsive dystonia (5 familial patients and 1 sporadic patient), aged 18 to 66 years. Data have been analyzed according to a graphic approach, calculating an influx constant for each region studied. We have also studied a seventh, clinically atypical, patient with juvenile dystonia-parkinsonism. Similar data have been calculated for a group of 10 healthy control subjects and 10 patients with Parkinson's disease. The 6 patients with typical dopa-responsive dystonia had a modest but significant reduction in the uptake of tracer into both caudate and putamen, which indicates a defect in the decarboxylation, vesicular uptake, and storage of [18F]dopa. This argues against the proposition that dopa-responsive dystonia is due to an inherited defect of tyrosine hydroxylase alone. In the atypical patient, however, we found a greater reduction of [18F]dopa uptake into both caudate and putamen, comparable with that in patients with Parkinson's disease.     

Frequency of GCH1 deletions in Dopa-responsive dystonia

We performed a systematic study on the frequency of point mutations and deletions of the gene GCH1 in dopa-responsive dystonia (DRD). A total of 136 dystonia patients were studied. Fifty of these had a sustained response to oral L-Dopa therapy (group 1: definite diagnosis of DRD), whereas the response to L-Dopa was incomplete or not tested in 86 patients (group 2: possible diagnosis of DRD). We found a GCH1 point mutation in 27 patients of group 1 (54%) and in four patients of group 2 (5%). Of these, nine single and one double mutation have not been described before. GCH1 deletions were detected in four patients of group 1 (8%) and in one patient of group 2 (1%). Among GCH1 point-mutation-negative patients with a definite diagnosis of DRD (group 1), the frequency of GCH1 deletions was 17% (4/23). We conclude that GCH1 deletion analysis should be incorporated into the routine molecular diagnosis of all patients with DRD with a sustained response to L-Dopa.     

Low frequency of Parkin, Tyrosine Hydroxylase, and GTP Cyclohydrolase I gene mutations in a Danish population of early-onset Parkinson''s Disease

Autosomal recessive Parkinson's disease (PD) with early-onset may be caused by mutations in the parkin gene (PARK2). We have ascertained 87 Danish patients with an early-onset form of PD (age at onset < or =40 years, or < or =50 years if family history is positive) in a multicenter study in order to determine the frequency of PARK2 mutations. Analysis of the GTP cyclohydrolase I gene (GCH1) and the tyrosine hydroxylase gene (TH), mutated in dopa-responsive dystonia and juvenile PD, have also been included. Ten different PARK2 mutations were identified in 10 patients. Two of the patients (2.3%) were found to have homozygous or compound heterozygous mutations, and eight of the patients (9.2%) were found to be heterozygous. A mutation has been identified in 10.4% of the sporadic cases and in 15.0% of cases with a positive family history of PD. One patient was found to be heterozygous for both a PARK2 mutation and a missense mutation (A6T) in TH of unknown significance. It cannot be excluded that both mutations contribute to the phenotype. No other putative disease causing TH or GCH1 mutations were found. In conclusion, homozygous, or compound heterozygous PARK2 mutations, and mutations in GCH1 and TH, are rare even in a population of PD patients with early-onset of the disease.     

Dopa-responsive dystonia and Tourette syndrome in a large Danish family

BACKGROUND: Guanosine triphosphate cyclohydrolase I (GTPCH) catalyzes the first step in the synthesis of tetrahydrobiopterin (BH4). Autosomal dominantly inherited defects in the GTPCH gene (GCH1) cause a form of dystonia that is responsive to treatment with levodopa (dopa-responsive dystonia [DRD]). OBJECTIVE: To investigate molecular and clinical aspects of DRD in a large Danish family. METHODS: For analysis of the GCH1 gene, a mutation-scanning method based on denaturing gradient gel electrophoresis (DGGE) was used. A novel mutation, X251R, was identified in the GCH1 gene of 2 distantly related Danish patients with DRD, one of whom also had Tourette syndrome (TS). Thirty-five additional family members were investigated for this mutation, and 16 of them underwent clinical neurological examination. RESULTS: A total of 18 patients were heterozygous for the X251R allele, 16 of whom had neurological complaints spanning from very mild parkinsonism to severe invalidism due to dystonia. Of 13 symptomatic heterozygotes who had been neurologically examined, 10 had signs of dystonia or parkinsonism. Sixteen of the heterozygotes were treated with levodopa, and 13 reported a treatment benefit. Three of the symptomatic heterozygotes had signs of TS. CONCLUSIONS: This study confirms the large variability in DRD symptoms and emphasizes the usefulness of molecular analysis for diagnosis and treatment of DRD. The presence of TS is suggested to be coincidental, though the development of TS-like symptoms due to mutations in GCH1 cannot be excluded.     

Biochemical and fluorodopa positron emission tomographic findings in an asymptomatic carrier of the gene for dopa-responsive dystonia

We report cerebrospinal fluid monoamine metabolite analyses and 6-[¹⁸F]fluoro-1-dopa positron emission tomography (FD-PET) from an asymptomatic carrier of the gene for dopa-responsive dystonia. Cerebrospinal fluid homovanillic acid, tetrahydrobiopterin, and neuropeterin concentrations were reduced in this man and in his affected children. His FD-PET was normal, as we have previously found in dopa-responsive dystonia. Neurological function and FD-PET may be normal despite marked abnormality in dopamine metabolism.     

Utility of MLPA in deletion analysis of <Emphasis Type="Italic">GCH1</Emphasis> in dopa-responsive dystonia

We applied multiple ligation-dependent probe amplification (MLPA) to patients from three families with characteristic dopa-responsive dystonia (DRD) but no base change in the gene GCH1. We found a complete deletion of GCH1 in affected members of family 1, and partial deletions in affected individuals of family 2 (exons 4-6) and of family 3 (exons 2-6). The findings were confirmed by quantitative real-time PCR. Our investigations demonstrate the utility of MLPA for routine deletion analysis of GCH1 in DRD patients with no sequence changes in this gene.     

Novel GCH-1 mutations and unusual long-lasting dyskinesias in Korean families with dopa-responsive dystonia

ObjectiveTo describe the long-term follow-up data of Korean patients with GTP cyclohydrolase (GTPCH) I deficient dopa-responsive dystonia (DRD) with novel mutations and unusual long-lasting dyskinesias.MethodsClinical features and genetic testing results of GCH1 from 19 patients that included 4 families who have been followed-up for up to 25 years were analyzed.ResultsGCH1 mutations were confirmed in all our symptomatic subjects including 3 novel point mutations. All the subjects except for one family had typical manifestations of autosomal dominant GTPCH-I deficient DRD including early childhood onset dystonia predominantly in the legs, marked diurnal variation, intact cognition, no other systemic symptoms, and excellent sustained response to levodopa. The one family who was the exception had two gene positive members of DRD and one with dopa-unresponsive cervical dystonia with negative GCH1 mutation. One family and a sporadic case had been reported as gene negative in a previous study, but they typically had preserved dopamine transporter binding and low neopterin levels in cerebrospinal fluids; thus, GCH-1 mutation had been highly suspected, which was now confirmed by repeating the genetic testing this time. An early childhood-onset patient developed choreiform dyskinesias right after administration of levodopa. The dyskinesia had lasted for more than 4 years regardless of the levodopa dosages and then subsided while maintaining levodopa.ConclusionThis report emphasizes the usefulness of the neopterin level in cerebrospinal fluids and dopamine transporter imaging in the differential diagnosis of DRD syndromes and a possible mechanism of levodopa-induced-dyskinesia in early childhood onset case.     

Dopa-responsive dystonia due to a large deletion in the GTP cyclohydrolase I gene

Although it is assumed that most patients with autosomal dominant dopa-responsive dystonia (DRD) have a GTP cyclohydrolase I dysfunction, conventional genomic DNA sequencing of the gene (GCH1) coding for this enzyme fails to reveal any mutations in about 40% of DRD patients, which makes molecular genetic diagnosis difficult. We found a large heterozygous GCH1 deletion, which cannot be detected by the usual genomic DNA sequence analysis, in a three-generation DRD family and conclude that a large genomic deletion in GCH1 may account for some "mutation-negative" patients with dominantly inherited DRD.     

Familial dopa-responsive cervical dystonia

The authors present four cases from two unrelated families with young-onset predominant cervical dystonia with a dramatic sustained response to levodopa. Onset age was 12 years (range 9 to 15). Additional symptoms included postural hand tremor and laryngeal dystonia. Genetic testing for GTP cyclohydrolase I, tyrosine hydroxylase, and sepiapterin reductase was negative. These cases may represent new forms of dopa-responsive dystonia. Levodopa is advisable in all patients with young-onset cervical dystonia.     

A novel missense mutation of the GTP cyclohydrolase I gene in a Korean family with hereditary progressive dystonia/dopa-responsive dystonia

Hereditary progressive dystonia with marked diurnal fluctuation/dopa-responsive dystonia (HPD/DRD) shows the considerable heterogeneity of clinical phenotypic expression and a dramatic sustained response to levodopa. The autosomal dominant HPD/DRD is caused by mutations in the gene coding GTP cyclohydrolase I (GCH I), the enzyme that catalyzes the first step in the biosynthesis of tetrahydrobiopterin. Previous studies suggested that normal [18F]Dopa positron emission tomography or [123I]beta-CIT single-photon emission computed tomography (SPECT) imaging, indicating intact structural integrity of nigrostriatal neurons, may be useful for differentiating HPD/DRD from clinically similar conditions such as juvenile Parkinson's disease with dystonia that have a considerably poorer prognosis. We here report a Korean family affected with HPD/DRD due to a novel missense mutation of the GCH I gene (T-->G mutation in exon 2), Met 137 Arg, which may change the conformation of the binding site of GCH I. The clinical features are considerably variable within the family. We documented normal striatal uptake of [123I]IPT, a dopamine transporter ligand with fast washout kinetics, in our patients by using SPECT. This method can be helpful in diagnosing HPD/DRD in uncertain cases.     

Dopa-responsive dystonia simulating spastic paraplegia due to tyrosine hydroxylase (TH) gene mutations

Spastic paraplegia is not widely recognized to occur in dopa-responsive dystonia (DRD). The authors found a compound heterozygote for novel mutations of the human tyrosine hydroxylase (TH) gene (TH). The patient was initially diagnosed as having spastic paraplegia, but responded completely to levodopa therapy. Exercise-induced stiffness in the patient's father, who had a TH deletion, also responded to levodopa. The data expand the clinical spectrum of TH deficiency and suggest that TH mutations may account for some patients with DRD simulating spastic paraplegia.     

Tyrosine hydroxylase,tryptophan hydroxylase,biopterin, and neopterin in the brain of anorexia nervosa

Summary The activities of tyrosine hydroxylase and tryptophan hydroxylase and contents of biopterin and neopterin were measured for the first time in various regions of human brain from a patient with anorexia nervosa (AN). In AN as compared with controls, tyrosine hydroxylase activity was markedly reduced in all brain regions analyzed, while tryptophan hydroxylase activity and biopterin content had a tendency to increase. Neopterin content did not change dramatically. The opposite changes of tyrosine hydroxylase and tryptophan hydroxylase suggest an imbalance between the activity of catecholaminergic neurons and that of serotonergic neurons, and may be related to pathogenesis of AN.     

Novel mutations in the guanosine triphosphate cyclohydrolase 1 gene associated with DYT5 dystonia

OBJECTIVES: To better understand the relationship between mutation of the guanosine triphosphate cyclohydrolase I (GCH1) gene and the etiology of DYT5 dystonia and to accumulate data on the mutation in the Japanese population for genetic diagnosis of the disease. SETTING: Japanese population. Patients Eight Japanese patients with suspected DYT5 dystonia were analyzed. Intervention Direct genomic sequencing of 6 exons of GCH1 was performed. MAIN OUTCOME MEASURES: For patients who did not exhibit any abnormality in the sequence analysis, the possibility of exon deletions was examined. In cases for which cerebrospinal fluid was available, the concentrations of neopterin and biopterin were measured as an index of GCH1 enzyme activity. RESULTS: In 2 patients, we found a new T106I mutation in exon 1 of GCH1, a position involved in the helix-turn-helix structure of the enzyme. In the third patient, we found a new mutation (a 15-base pair nucleotide deletion) in exon 5 that may cause a frameshift involving the active site. In the fourth patient, we detected a known nucleotide G>A substitution in the splice site of intron 5, which has been reported to produce exon 5-skipped messenger RNA. The concentrations of both neopterin and biopterin in the cerebrospinal fluid of the third and fourth patients were markedly lower than the normal range, indicating that the GCH1 enzyme was functionally abnormal in these mutations. Gene dosage analysis showed that the fifth patient had a deletion of both exon 3 and exon 4, whereas the sixth patient had a deletion of exon 3. CONCLUSIONS: We found several novel, as well as known, GCH1 mutations in Japanese patients with DYT5 dystonia. In some of them, the GCH1 enzyme activity was proved to be impaired.