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1
Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland.
a Author for correspondence. Fax 41-1-266-7169; e-mail
Nenad.Blau{at}kispi.unizh.ch.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Neopterin and biopterin production and GTP cyclohydrolase I (GTPCH) activity were measured in cytokine-stimulated fibroblasts; 6-pyruvoyltetrahydropterin synthase (PTPS), sepiapterin reductase (SR), and dihydropteridine reductase (DHPR) activities were measured in unstimulated fibroblasts. We examined 8 patients with DRD, 3 with autosomal recessive GTPCH deficiency, 7 with PTPS deficiency, 3 with DHPR deficiency, and 49 controls (35 fibroblast and 14 amniocyte samples).
Results: Fibroblasts from patients with DRD and autosomal recessive GTPCH deficiency showed reduced GTPCH activity (15.4% and 30.7% of normal activity, respectively) compared with controls (P <0.001). Neopterin production was very low and biopterin production was reduced in both disorders. PTPS- and DHPR-deficient cells showed no enzyme activities; in PTPS deficiency the pattern of pterin production was typical (neopterin, 334–734 pmol/mg; controls, 18–98 pmol/mg; biopterin, 0 pmol/mg; controls, 154–303 pmol/mg). Reference values of all enzyme activities and pterin production were measured in fibroblasts and also in amniocytes for prenatal diagnosis.
Conclusions: Cultured skin fibroblasts are a useful tool in the diagnosis of BH4 deficiencies. Intracellular neopterin and biopterin concentrations and GTPCH activity in cytokine-stimulated fibroblasts are particularly helpful in diagnosing patients with DRD.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (1). Phenylalanine-3-hydroxylase is responsible for
hydroxylation of phenylalanine to tyrosine in the liver.
Tyrosine-4-hydroxylase and tryptophan-5-hydroxylase are the
rate-limiting enzymes in the biosynthesis of the neurotransmitters
dopamine and serotonin. BH4 is also a cofactor
for the different forms of nitric oxide synthase.
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GTP cyclohydrolase I (GTPCH; EC 3.5.4.16) catalyzes the first and
rate-limiting step in the BH4 pathway. It is
expressed in peripheral tissues as well as in the brain, and its
expression can be induced at the transcriptional level in T
lymphocytes, macrophages, and fibroblasts by cytokines such as
interferon-
(IFN-) and tumor necrosis factor-
(TNF-)
(2)(3). The enzyme converts GTP to the
intermediate 7,8-dihydroneopterin triphosphate (Fig. 1
).
6-Pyruvoyltetrahydropterin synthase (PTPS; EC 4.6.1.10) is the second
enzyme in the biosynthesis of BH4. It is
constitutively expressed in many cell types, including fibroblasts, and
catalyzes the synthesis of 6-pyruvoyltetrahydropterin from
7,8-dihydroneopterin triphosphate. The last step in the biosynthesis of
BH4 is catalyzed by sepiapterin reductase (SR; EC
1.1.1.153), which reduces 6-pyruvoyltetrahydropterin to
BH4. Two enzymes are responsible for the
regeneration of BH4 following oxidation by the
amino acid mono-oxygenases: pterin-4-carbinolamine dehydratase (EC
4.2.1.96) and dihydropteridine reductase (DHPR; EC 1.6.99.7; Fig. 1
).
At least DHPR is widely distributed in animal tissues and
constitutively expressed in fibroblasts (4).
Autosomal recessive mutations in the genes coding for GTPCH, PTPS, or DHPR are responsible for BH4 deficiencies (5)(6)(7). Most of these deficiencies are characterized by neonatal hyperphenylalaninemia, developmental delay, progressive neurological deterioration, hypokinesia, hypersalivation and drooling, swallowing difficulties, truncal hypotonia, increased limb tone, myoclonus, and temperature instability, with onset in the first months of life (8). More than 400 patients are tabulated in the international database (7).
Dopa-responsive dystonia (DRD) is a clinical syndrome characterized by childhood-onset dystonia (usually before 12 years of age), with dramatic clinical normalization that is sustained with low doses of L-Dopa (9). The dystonia typically affects the legs, causing gait disturbance and postural instability. Signs and symptoms usually are progressive and worsen later in the evening or after exertion (diurnal fluctuation) (10). Elements of Parkinsonism (rigidity, bradikynesia, and rest tremor) commonly occur, especially in adulthood (11). Early motor development usually is normal, and hyperphenylalaninemia is absent. Recent studies have demonstrated that DRD is caused by autosomal dominant mutations in the coding region of the GCH1 gene, which encodes for GTPCH (12), but only in 40–50% of cases (13). Reduced penetrance and gender-related vulnerability are reported, with females being more affected than males (2.5:1) (14). The clinical phenotypic expression is quite diverse (15), and many cases appear to be sporadic (16).
Today, the diagnosis of BH4 deficiencies is based on neonatal screening for phenylketonuria and urinary pterin analysis (17). DRD is diagnosed mainly by clinical signs and symptoms and by response to L-Dopa/Carbidopa treatment. The examination of pterins and neurotransmitter metabolites in cerebrospinal fluid (CSF) requires an invasive procedure, but it is the only way to make a reliable diagnosis of DRD (10)(18). Definitive diagnosis of BH4 deficiencies is achieved by enzyme activity determinations and DNA analysis (8).
PTPS and DHPR activities can be measured in liver biopsies (19)(20), erythrocytes (21)(22), and fibroblasts (23)(24), whereas GTPCH is measurable in liver biopsies (25). In mononuclear blood cells, GTPCH can be induced by phytohemagglutinin (26), and reference values have been reported recently by Hibiya et al. (27). However, this test is quite complex and must be performed within 20 h after sample collection (27). In previous studies, fibroblasts were used for indirect measurement of pterin metabolism (28), based on the induction of GTPCH expression with cytokines, but no data are available about nonpathological and pathological values. The possibility of measuring GTPCH activity in cytokine-stimulated fibroblasts was shown by Werner et al. (29). In the present study, we used a similar stimulation procedure for measurement of both pterins and GTPCH activity. To our knowledge, fibroblasts have not been systematically tested for the other BH4-metabolizing enzymes. Cultured skin fibroblasts are easily used and practical for the diagnosis of many metabolic disorders. The samples can be transported at room temperature and stored for long periods after early passages for later analysis.
The aim of the present study was to demonstrate the utility of cultured skin fibroblasts as a diagnostic tool for DRD and BH4 deficiencies. Particularly for DRD, the enzyme activity measurement is essential for final diagnosis. BH4 metabolism was studied in fibroblasts from a large number of controls and in patients with different forms of BH4 deficiency.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Control value biopsies were obtained from 17 subjects (10 males, 7 females) between birth and 1 year of age and 18 subjects (11 males, 7 females) between 1 and 45 years of age. No neurological abnormalities were detected in any of the controls. To establish reference values for prenatal diagnosis, 14 amniocyte samples collected between 15 and 18 weeks of gestational age were also studied. The activities of all of the BH4-metabolizing enzymes, as well as neopterin and biopterin production after stimulation with cytokines, were measured in all samples.
The procedures used were in accordance with the current revision of the Helsinki Declaration of 1975.
materials
Sephadex G-50 Nick spin columns were from Pharmacia. DMEM,
AmnioMax C-100 medium, trypsin/EDTA solution, antibiotics, and fetal
calf serum were purchased from Life Technologies. GTP lithium salt,
recombinant human IFN-, and TNF- were from Sigma. Calf intestine
alkaline phosphatase, NADH, NADPH, DHPR, peroxidase, leupeptin, and
pepstatin were from Roche. Falcon cell culture plasticware was from
Becton-Dickinson. -Globulin and the protein assay dye reagent were
from Bio-Rad. Ultrafree-MC filters with PL10 membrane were from
Millipore.
Pteridine derivatives (6,7-dimethyltetrahydropterin and sepiapterin) were from Schircks Laboratories. Dihydroneopterin triphosphate was prepared enzymatically as described previously (30), but using recombinant GTPCH (Thöny B, Blau N. Enzymatic reactor for synthesis of dihydroneopterin triphosphate. Manuscript in preparation). Recombinant SR was purified in our laboratory (Thöny B, unpublished work). All other chemicals were from Fluka.
cell culture and treatment with cytokines
Fibroblasts were cultured in DMEM containing 100 mL/L fetal
bovine serum, 50 kilounits/L penicillin, and 50 mg/L
streptomycin. Amniocytes were cultured in AmnioMax
C-100 medium. Cells were passaged by trypsinization and studied at low
passage numbers (3–12). All cells were Mycoplasma negative,
as checked by the Hoechst staining method (31). Cultures
were kept at 37 °C in humidified air containing 5%
CO2.
To induce the expression of GTPCH, confluent cell monolayers in
78-cm2 plates were stimulated with recombinant
human IFN- and TNF- at concentrations of 250 and 100 kilounits/L,
respectively, in fresh medium. After incubation for 24 h, cells
were harvested by trypsinization, washed with phosphate-buffered
saline, and immediately lysed for neopterin and biopterin measurement
and for GTPCH activity assay. PTPS, DHPR, and SR are constitutively
expressed in fibroblasts; therefore, their activities were tested in
unstimulated cells. Confluent cells in 78-cm2
plates were harvested, washed with phosphate-buffered saline, and kept
at -80 °C until analyzed for PTPS, DHPR and SR.
measurement of neopterin and biopterin production in cell extracts
Cytokine-stimulated cells from one confluent
78-cm2 plate were lysed in 150 µL of 50 mmol/L
Tris-HCl (pH 7.4) containing 1 mmol/L EDTA and 1 mmol/L
dithioerythritol by freezing and thawing six times. Lysate was
centrifuged at 15 000g, and 100 µL of the supernatant was
oxidized with 6 µL of 1 mol/L HCl and 20 µL of 10 g/L iodine
solution for 15 min at room temperature in the dark. Oxidation was
stopped by the addition of 20 µL of 10 g/L ascorbic acid (freshly
prepared). Ten microliters of buffer (1 mol/L Tris-HCl, pH 9.6,
containing 80 mmol/L MgCl2) was added to the
sample to adjust to pH 8.0–9.0. Dephosphorylation of neopterin
triphosphate was achieved by hydrolysis with alkaline phosphatase (16.8
U) for 60 min at 37 °C. The reaction was stopped by acidification to
pH 2.0 with 7 µL of 2 mol/L HCl. The sample (final volume, 175 µL)
was subsequently deproteinized through an Ultrafree-MC filter and
analyzed by HPLC (32). The intracellular concentrations of
neopterin and biopterin after 24-h stimulation with cytokines are
expressed as pmol per mg of protein.
gtpch assay
The assay was performed as described previously by Hatakeyama and
Yoneyama (33), with some modifications. The assay monitors
the conversion of the substrate GTP under saturating conditions to
neopterin triphosphate, which is detected as neopterin, the oxidized
and dephosphorylated product (Fig. 1
).
Fibroblasts were analyzed immediately after 24-h incubation with cytokines. Cells from one confluent 78-cm2 plate were lysed in 200 µL of freshly prepared homogenization buffer (50 mmol/L Tris-HCl, pH 7.5, containing 0.1 mol/L KCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.2 mmol/L phenylmethylsulfonyl fluoride, 1 µmol/L leupeptin, and 1 µmol/L pepstatin) by freezing and thawing six times and were subsequently centrifuged at 15 000g for 5 min. A 150-µL aliquot of the supernatant was desalted on a spin column (Nick spin column, Sephadex G50; Pharmacia Biotech), and 50 µL of the filtrate was added to 148 µL of reaction buffer (50 mmol/L Tris-HCl, pH 7.5, containing 0.1 mol/L KCl and 1 mmol/L EDTA) and 2 µL of 100 mmol/L GTP. The mixture was divided into two 100-µL portions; one was incubated for 60 min at 37 °C, whereas the other was immediately oxidized (blank with cell extract). A blank without cell extract (25 µL of homogenization buffer added to 1 µL of 100 mmol/L GTP and to 74 µL of reaction mixture) was incubated together with the sample. The reaction was stopped by cooling the sample on ice and adding 10 µL of oxidizing solution (5 g/L iodine and 10 g/L potassium iodide in 1 mol/L HCl). After oxidation in the dark for 60 min, the reaction was stopped by adding 10 µL of 20 g/L ascorbic acid (freshly prepared). The mixture was adjusted to pH 8.5 by adding 14 µL of 1 mol/L NaOH, and the sample was incubated with 20 µL of alkaline phosphatase solution (300 kU/L calf intestine alkaline phosphatase in 0.1 mol/L Tris-HCl, pH 8.0, containing 1 mmol/L MgCl2 and 0.1 mmol/L ZnCl2) for 60 min at 37 °C. Neopterin was measured, after deproteinization through an Ultrafree-MC filter, by HPLC (32). One unit of GTPCH produces 1 µmol of neopterin per minute at 37 °C.
ptps assay
The PTPS activity was measured in extracts from unstimulated
cells. The assay was modified according to Shintaku et al.
(21). The unstable intermediate metabolite formed from
dihydroneopterin triphosphate is converted to BH4
by SR in the presence of NADPH. DHPR activity is necessary to stabilize
the formation of BH4. BH4
is then measured as the oxidized product, biopterin.
Unstimulated cells from one confluent 78-cm2 plate were suspended in 150 µL of lysis buffer (10 mmol/L Tris-HCl, pH 7.4, containing 10 mL/L Triton X-100) and lysed by freezing and thawing six times. After centrifugation at 15 000g for 5 min, 50 µL of the supernatant was added to 60 µL of reaction mixture containing 100 mmol/L Tris-HCl, pH 7.4, 10 mmol/L MgCl2, 1 mmol/L NADPH, 1 mmol/L NADH, 3 mU of SR, 220 mU of DHPR, and 60 µmol/L dihydroneopterin triphosphate in a final volume of 110 µL. A 50-µL aliquot was incubated for 120 min at 37 °C; another 50 µL was used as a blank. A blank without cell extract, containing 50 µL of lysis buffer and 60 µL of reaction mixture, was incubated in the same run. The reaction was stopped by cooling the samples on ice and adding 15 µL of 300 g/L trichloroacetic acid for protein precipitation, followed by centrifugation (5 min at 15 000g) and oxidation. Oxidation was performed by incubation of 50 µL of the supernatant with 10 µL of 10 g/L iodine for 60 min at room temperature in the dark. The same procedure was used for the blanks. After oxidation, 15 µL of 10 g/L ascorbic acid was added to destroy excess iodine. Samples were diluted (1:4 by volume) with water and heated at 60 °C for 2 h before being deproteinized through an Ultrafree-MC filter and analyzed by HPLC (32). One unit of PTPS produces 1 µmol of biopterin per minute at 37 °C.
sr assay
The activity was assayed according to the method described by
Ferre and Naylor (34) with some modifications. The assay
monitors the conversion of sepiapterin to BH2,
which is then measured as the oxidized product, biopterin.
Unstimulated cells from one confluent 78-cm2 plate were suspended in 1 mL of lysis buffer (0.1 mol/L potassium phosphate buffer, pH 6.4, containing 0.15 mol/L KCl, 0.68 mol/L glycerol, and 2.5 mmol/L EDTA), lysed by freezing and thawing six times, and centrifuged for 5 min at 15 000g. A 20-µL portion of the supernatant was incubated in a freshly prepared reaction mixture (final volume, 50 µL) containing 0.1 mol/L potassium phosphate buffer, pH 6.4, 125 µmol/L sepiapterin, and 250 µmol/L NADPH at 37 °C for 30 min in the dark. A blank with cell extract was immediately oxidized, and a blank without cell extract (20 µL of lysis buffer in 50 µL of reaction mixture) was incubated with the samples. The reaction was stopped by adding 10 µL of oxidizing solution (5 g/L iodine and 10 g/L potassium iodide in 1 mol/L HCl) and maintained at room temperature in the dark for 30 min. Excess iodine was removed by adding 10 µL of 10 g/L ascorbic acid solution (freshly prepared). The sample was then deproteinized through an Ultrafree-MC filter and analyzed by HPLC (32). One unit of enzyme activity produces 1 µmol of biopterin per minute at 37 °C.
dhpr assay
The activity of this enzyme was assayed, as described previously
(24), by monitoring the oxidation of NADH during the
reduction of 6,7-dimethyldihydropterin (quinonoid isomer) to
6,7-dimethyltetrahydropterin, catalyzed by DHPR. The pterin derivative
6,7-dimethyltetrahydropterin was oxidized in situ by peroxidase in the
presence of H2O2 to
q-6,7-dimethyldihydropterin and thus was recycled for the DHPR
reaction.
Unstimulated fibroblasts from three confluent 78-cm2 plates were suspended in 450 µL of lysis buffer (5 mmol/L Tris-HCl, pH 7.4, containing 0.1 mol/L KCl), lysed by freezing and thawing six times, and centrifuged at 15 000g for 20 min. The supernatant was then analyzed for protein concentration, and an amount corresponding to 120 µg of protein was incubated in the assay. The assay mixture contained 8.8 mmol/L H2O2, 1 mmol/L NADH, 25 kU/L peroxidase, and reaction buffer (50 mmol/L Tris-HCl, pH 7.0) in a final volume of 1 mL. After a 5-min incubation at 25 °C and before the substrate was added, the absorbance at 340 nm of the samples and blanks (without cell extract) was measured. Subsequently, 15 µL of substrate (1 mmol/L 6,7-dimethyltetrahydropterin) was added and mixed, and the absorbance was measured at the same wavelength (340 nm). The consumption of NADH was measured against corresponding blanks at 340 nm for 5 min. One unit of DHPR oxidizes 1 µmol of NADH per minute at 25 °C.
protein measurement
Protein concentrations in cell lysates were determined by the
Bradford spectrophotometric method (35) using -globulin
as a calibrator. The activities of the various enzymes are expressed as
units per mg of protein.
statistical analysis
Normal values of neopterin, biopterin, and enzyme activities for
controls were expressed as 5th, 50th, and 95th percentiles (Table 1
). Medians and minimum and maximum concentrations of the same
analytes were used for pathological values because of the limited
number of samples (Table 2
). Statistical differences between multiple groups (neopterin
and biopterin concentrations in controls and patients with different
disorders; GTPCH activity in controls and patients with DRD and GTPCH
deficiency) were tested using a one-way ANOVA with pairwise
comparison according to Tukey. The unpaired Student t-test
was used to compare results between two main control groups (amniocytes
vs fibroblasts, males vs females, fibroblasts 0–1 year vs fibroblasts
>1 year) and between particular enzyme activities in controls and
patients with different disorders. Differences were considered
significant when P values were <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). No differences in the production of neopterin (males, 30–94
pmol/mg; females, 17–99 pmol/mg) and biopterin (males, 189–301
pmol/mg; females, 153–327 pmol/mg) were found between fibroblasts from
males (n = 21) and females (n = 14). GTPCH activity was very
low in amniocytes but increased in fibroblasts collected after birth
(P <0.01). Fibroblast GTPCH activity was higher in females
than in males (females, 1.4–7.3 µU/mg; males, 1.4–3.9 µU/mg;
P = 0.016). No significant difference was observed at
different ages (0–1 year, 1.7–4.9 µU/mg; >1 year, 1.3–7.3
µU/mg; P = 0.97; activity 0–10 years vs activity
>10 years, P = 0.74).
reference values for ptps, sr, and dhpr activities in unstimulated
cells
PTPS activity was higher in amniocytes than in fibroblasts
(P <0.001; Table 1
). A decrease in enzyme activity was
observed in cells collected after the first year of age (0–1 year,
0.5–1.7 µU/mg; >1 year, 0.4–0.7 µU/mg; P =
0.014). No differences were observed in other age groups after 1 year.
Enzyme activities in males (0.4–1.0 µU/mg) vs females (0.4–1.8
µU/mg) showed no significant differences. SR activity was similar in
cells collected before and after birth, as well as in males and females
(males, 96–202 µU/mg; females, 120–155 µU/mg) and also in
different age groups. DHPR activity was also similar in amniocytes and
fibroblasts, as well as in males and females (males, 5–7.8 mU/mg;
females, 4.2–8.9 mU/mg). A decrease in activity was observed after 1
year of age (0–1 year, 5.4–8.9 mU/mg; >1 year, 4.2–7.0 mU/mg;
P = 0.03). No differences were observed in other age
groups after 1 year. There were no significant increases in PTPS, SR,
and DHPR activities after stimulation with cytokines (data not shown).
measurement of neopterin and biopterin production and enzyme
activities in fibroblasts from patients
Neopterin and biopterin concentrations were significantly
different between controls and patients (P <0.001; Table 2
). Cytokine-stimulated GTPCH-deficient cells (autosomal recessive
form, three cases) showed extremely low concentrations of neopterin and
biopterin (Fig. 2
) and a median enzyme activity of 0.8 µU/mg of protein (30.7%
of the 50th percentile for normal activity). Cells from DRD patients
(eight cases) also showed reduced neopterin and biopterin
concentrations compared with controls (Fig. 2
). In both diseases, GTPCH
activity was lower than in controls (P <0.001). In DRD
patients, the median enzyme activity was 0.4 µU/mg of protein (15.4%
of the 50th percentile for normal activity), ranging between 0.1
(3.8%) and 0.6 µU/mg (23%). GTPCH activity in the autosomal
recessive form was higher than in the autosomal dominant form (GTPCH
deficiency vs DRD, P <0.01). PTPS-deficient cells (seven
cases) contained extremely high concentrations of neopterin (median,
475 pmol/mg; range, 334–734 pmol/mg), and no measurable biopterin
(Fig. 2
) after stimulation with cytokines for 24 h. Enzyme
activity, measured in unstimulated cells, was always below the
detection limit (<0.05 µU/mg, 7% of 50th percentile for controls).
DHPR-deficient cells (three cases) showed intracellular pterin
concentrations within reference values after stimulation (for
neopterin, median 49 pmol/mg; range 29–81 pmol/mg; for biopterin,
median, 179 pmol/mg; range, 99–181 pmol/mg; Fig. 2
), with biopterin
concentrations significantly lower than in controls (P
<0.001). Enzyme activity was not detectable in unstimulated
fibroblasts from the same patients (<0.3 mU/mg, 5% of 50th percentile
for controls).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DRD is diagnosed mainly by the typical clinical signs and symptoms and by the responsiveness to therapy with L-Dopa/Carbidopa. Hyperphenylalaninemia usually is not present in these patients; however, the oral phenylalanine loading test is consistent with a partial deficiency of BH4 in the liver. Plasma phenylalanine/tyrosine ratios increase significantly 4–6 h after the challenge (38). Unfortunately, false-positive results with this test are possible; in fact, heterozygous phenylketonuric subjects show the same abnormal phenylalanine/tyrosine profiles (39), whereas a small number of genetically confirmed DRD subjects showed no abnormalities with this test (40). A lumbar puncture for CSF examination is invasive but most informative for the diagnosis of both recessive BH4 deficiencies (41) and DRD (10). Low CSF concentrations of homovanillic acid and 5-hydroxyindoleacetic acid, metabolites of dopamine and serotonin, respectively, are found in all variants of BH4 deficiencies. Furthermore, both neopterin and biopterin are very low in GTPCH deficiency, neopterin is high and biopterin low in PTPS deficiency, and biopterin concentrations are very high in DHPR deficiency (8)(41). CSF examinations in DRD patients revealed low concentrations of homovanillic acid, indicating a lack of dopamine synthesis, and low neopterin and biopterin concentrations are consistent with a GTPCH deficiency (10).
The final diagnosis of BH4 deficiencies is achieved by enzyme activity determinations and, with some limitations, by detection of mutations. PTPS and DHPR activities are measurable in many different tissues and cells such as erythrocytes (21)(22), fibroblasts (23)(24), and liver (19)(20). Dried blood spots are routinely used for DHPR deficiency screening among hyperphenylalaninemic newborns (22). Measurement of GTPCH activity is rather difficult because this enzyme is not expressed in blood cells and fibroblasts. Blau et al. (26) described an assay in phytohemagglutinin-stimulated mononuclear blood cells that was used to measure reduced activity in heterozygous and homozygous GTPCH subjects, and subsequently in DRD patients (12). The test must be performed with fresh blood samples, within 20 h after sample collection (27). Bezin et al. (42) suggested that the stimulation of lymphoblasts could lead to incorrect determinations of the actual activity of GTPCH in DRD patients.
Because not all GTPCH-deficient DRD patients have GCH1
mutations in the coding region or in the splice sites, which are
detectable by the current genomic DNA sequencing of GCH1
(43), and because of the high occurrence of sporadic
mutations in this gene (14), DNA testing for the autosomal
dominant DRD is not suitable for routine clinical practice. Suzuki et
al. (44) reported that in 40% of DRD patients no mutation
was found in the GCH1 gene but that some of them showed
reduced GTPCH activity in phytohemagglutinin-stimulated mononuclear
blood cells. Therefore, measurement of enzyme activity is extremely
important for the diagnosis of DRD. Recent studies demonstrated that
cytokine-stimulated fibroblasts may be used for investigating
BH4 pathway integrity (28), by
measuring intracellular neopterin and biopterin production patterns,
and for GTPCH activity measurement (29). Here, we adapted
these methods to investigate in cytokine-stimulated fibroblasts both
pterin production patterns and GTPCH activity, with a diagnostic
purpose. We first determined the reference values of intracellular
neopterin and biopterin in fibroblasts and amniocytes after stimulation
with cytokines. Under these conditions, amniocytes appeared to produce
rather low amounts of pterins, consistent with their low GTPCH activity
(Table 1
). Therefore, these cells are not suitable for prenatal
diagnosis of GTPCH deficiency. On the other hand, prenatal diagnosis of
PTPS and DHPR deficiency should be possible because the activities of
these two enzymes are well detectable in amniocytes (Table 1
).
We observed that cells collected in the first year of life showed higher PTPS and DHPR activities than cells collected at a later age. GTPCH activity was slightly but not significantly higher when cells were collected during the first year of life. Because Hibiya et al. (27) reported a higher GTPCH activity in mononuclear blood cells in the first 10 years of life, we compared fibroblasts of these two control groups and found no significant difference. Only GTPCH activity was different between males and females, with higher activities in females. In contrast, lower activities in females were reported by Ichinose et al. (12), and no difference was found by Hibiya et al. (27) in mononuclear blood cells. The analysis of a larger collection of controls is probably needed to better define these differences in fibroblasts; however, this is not of diagnostic importance.
The study of pathological samples allowed us to define the utility of
cultured skin fibroblasts in the diagnosis of BH4
defects and DRD. Except for SR, for which no patients have as yet been
reported, all the studied enzymes exhibited very low activities in
patient samples compared with controls (Table 2
). Indeed, PTPS and DHPR
activities in unstimulated fibroblasts from the corresponding patients
were below the detection limit (<7% and <5% of the 50th percentile
of normal activity, respectively). PTPS deficiency was characterized by
a typical pattern of pterin production in stimulated fibroblasts, with
high neopterin concentrations and no measurable biopterin. The same
pattern was observed in urine and CSF (8). DHPR-deficient
patients showed normal intracellular neopterin concentrations after
stimulation, whereas biopterin concentrations were lower than in
controls. Milstien et al. (28) suggested that the possible
pattern of pterin production in these cells could be characterized by
increased neopterin because of the lack of feedback inhibition of GTPCH
by BH4, and reduced biopterin because of the fast
transport rates of the oxidized forms of pterins out of the cells. This
hypothesis would explain the differences of pterin patterns between the
cell extracts and the extracellular fluids (plasma, urine, CSF), where
increased biopterin is characteristic of the disease (41).
GTPCH activity was lower in stimulated fibroblasts from patients with
the autosomal dominant form than in those from patients with the
autosomal recessive form. Previous measurements in
phytohemagglutinin-stimulated mononuclear blood cells showed a
reduction of GTPCH activity in DRD patients to <20% (12),
which was comparable to our finding, and a reduction of activity to
<3% in autosomal recessive GTPCH deficiency (26), in
contrast to our results in stimulated fibroblasts (Table 2
). A
dominant-negative effect of the mutated subunit on the activity of the
wild-type subunit in DRD was suggested by Hirano and Ueno
(45). Indeed, cotransfection experiments with two mutant
GTPCH alleles, identified in DRD patients, demonstrated that mutant
proteins inhibit the activity of the wild-type enzyme. In contrast,
cotransfection of the mutant allele found in a patient with autosomal
recessive GTPCH deficiency failed to inhibit the wild-type activity,
producing much higher GTPCH activity (46). The molecular
mechanism of this dominant-negative effect and the different phenotype,
which is more severe in recessive GTPCH deficiency despite higher
enzyme activity in vitro, remain to be explained. Recently, Suzuki et
al. (44) reported that a reduction in the amount of
enzymatic protein could be responsible for the lower GTPCH activity in
DRD compared with the autosomal recessive GTPCH deficiency. A
dominant-negative effect might still be acting at an earlier stage, at
either the translational or the transcriptional level.
Regardless, GTPCH activity measurement in cytokine-stimulated
fibroblasts was diagnostic for both disorders.
In conclusion, skin cultured fibroblast assays are confirmatory for all suspected BH4 deficiencies. Our studies now provide for the first time reference values for direct and indirect (intracellular neopterin and biopterin) estimation of enzyme activity. The study of BH4 metabolism is particularly useful in DRD patients because it allows an etiologic diagnosis when other tests are inconclusive.
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Acknowledgments |
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Footnotes |
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, interferon-; TNF-, tumor necrosis factor-; PTPS, 6-pyruvoyltetrahydropterin synthase; SR, sepiapterin reductase; DHPR, dihydropteridine reductase; DRD, Dopa-responsive dystonia; and CSF, cerebrospinal fluid. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Biochem J 1989;262:861-866.[Web of Science][Medline]
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  M von Mering, H Gabriel, T Opladen, G F Hoffmann, and A Storch A novel mutation (c.64_65delGGinsAACC [p.G21fsX66]) in the GTP cyclohydrolase 1 gene that causes Segawa disease J. Neurol. Neurosurg. Psychiatry, February 1, 2008; 79(2): 229 - 229. [Full Text] [PDF]
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 J. D. Widder, W. Chen, L. Li, S. Dikalov, B. Thony, K. Hatakeyama, and D. G. Harrison Regulation of Tetrahydrobiopterin Biosynthesis by Shear Stress Circ. Res., October 12, 2007; 101(8): 830 - 838. [Abstract] [Full Text] [PDF]
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 K. Hyland Inherited Disorders Affecting Dopamine and Serotonin: Critical Neurotransmitters Derived from Aromatic Amino Acids J. Nutr., June 1, 2007; 137(6): 1568S - 1572S. [Abstract] [Full Text] [PDF]
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J L K Van Hove, J Steyaert, G Matthijs, E Legius, P Theys, R Wevers, A Romstad, L B Moller, K Hedrich, D Goriounov, et al. Expanded motor and psychiatric phenotype in autosomal dominant Segawa syndrome due to GTP cyclohydrolase deficiency J. Neurol. Neurosurg. Psychiatry, January 1, 2006; 77(1): 18 - 23. [Abstract] [Full Text] [PDF]
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 N. Franscini, E. B. Bachli, N. Blau, M. Fischler, R. B. Walter, A. Schaffner, and G. Schoedon Functional Tetrahydrobiopterin Synthesis in Human Platelets Circulation, July 13, 2004; 110(2): 186 - 192. [Abstract] [Full Text] [PDF]
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 N. Blau, L. Bonafe, I. Krageloh-Mann, B. Thony, L. Kierat, M. Hausler, and V. Ramaekers Cerebrospinal fluid pterins and folates in Aicardi-Goutieres syndrome: A new phenotype Neurology, September 9, 2003; 61(5): 642 - 647. [Abstract] [Full Text] [PDF]
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 L. Elzaouk, W. Leimbacher, M. Turri, B. Ledermann, K. Burki, N. Blau, and B. Thony Dwarfism and Low Insulin-like Growth Factor-1 Due to Dopamine Depletion in Pts-/- Mice Rescued by Feeding Neurotransmitter Precursors and H4-biopterin J. Biol. Chem., July 18, 2003; 278(30): 28303 - 28311. [Abstract] [Full Text] [PDF]
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N. Nardocci, G. Zorzi, N. Blau, E. Fernandez Alvarez, M. Sesta, L. Angelini, M. Pannacci, F. Invernizzi, and B. Garavaglia Neonatal dopa-responsive extrapyramidal syndrome in twins with recessive GTPCH deficiency Neurology, January 28, 2003; 60(2): 335 - 337. [Abstract] [Full Text] [PDF]
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 A. H. Nemeth The genetics of primary dystonias and related disorders Brain, April 1, 2002; 125(4): 695 - 721. [Abstract] [Full Text] [PDF]
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L. Bonafe, N. Blau, A. P. Burlina, A. Romstad, F. Guttler, and A. B. Burlina Treatable neurotransmitter deficiency in mild phenylketonuria Neurology, September 11, 2001; 57(5): 908 - 911. [Abstract] [Full Text] [PDF]
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and 
, respectively),
and range (bars)] in cytokine-stimulated fibroblasts
from controls (n = 35), DRD patients (n = 8), autosomal
recessive GTPCH-deficient patients (n = 3), PTPS-deficient
patients (n = 7), and DHPR-deficient patients (n = 3).