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Dominant negative effect of GTP cyclohydrolase I mutations in dopa-responsive hereditary progressive dystonia.

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Dominant Negative Effect of GTP
Cyclohydrolase I Mutations in DopaResponsive Hereditary Progressive Dystonia
Makito Hirano, MD,*t$ Takehiko Yanagihara, MD,* and Satoshi Ueno, MDt
Hereditary progressive dystonia (HPD) is caused by the mutant gene encoding GTP cyclohydrolase I (GCH). The clinical
presentation of this disease varies considerably, and many cases appear to be sporadic. We have previously proposed that
this clinical variation may be due to digetential expression of the mutant and normal GCH mRNA, presumably at the
protein level. To provide support for this proposal, we studied a new Japanese family with HPD, in which 2 members were
acid resheterozygous for an exon-skipping mutation. This mutation produced truncated GCH, which s h e d 180-&0
idues at the amino terminus of the normal enzyme (GCH180). An affected heterozygote had a higher mutandnormal
mRNA ratio than an unaffeaed heterozygote, consistent with our previous finding in the HPD M y with GCHl14. A
further study, using coexpression of the mutant with wild-type GCH in COS-7 cells, showed that three mutant GCHs
inactivated the normal enzyme. GCH114 was most effective in enzyme inactivation, which was followed by GCH180 and
a normally occurring mutant GCH209. These results suggested that the dominant negative a c t of a mutant GCH on the
normal enzyme might be one of the molecular mechanisms determining the heterogeneity of clinical phenotypes of HPD.
Hirano M, Yanagihara T, Ueno S. Dominant negative effect of GTP cyclohydrolase I mutations in
dopa-responsive hereditary progressive dystonia. Ann Neurol 1998;44:365-37 1
Hereditary progressive dystonia (HPD) with marked diurnal fluctuationidopa-responsive dystonia is a disease
with childhood onset characterized by postural dystonia
that pronouncedly responds to levodopa. Recent studies
have demonstrated that HPD is caused by mutations in
the gene encoding GTP cyclohydrolase I (GCH), which
catalyzes a rate-limiting step in the biosynthesis of tetrahydrobiopterin, an essential cofactor of tyrosine hydroxylase.'-' One of the puzzling aspects of HPD is the considerable diversity of clinical phenotypic expression, where
many cases appear to be sporadic.'-7 To explain the diversity, we have previously proposed that a relative increase in the mutantlnormd GCH mRNA ratio and presumably the mutant/normal protein ratio are responsible
for the heterogeneous phenotypic manifestations, where
an increased number of mutant subunit peptides may interact with wild-type peptides and lead to a higher proportion of nonhctional multimers, and therefore a more
severe clinical p h e n ~ t y p e .To
~ , ~support this hypothesis, it
is crucial to demonstrate that mutations in the GCH gene
inhibit the normal enzyme activity.
Recently we identified, in a new Japanese family, a
patient with HPD and his unaffected father who were
heterozygous for a novel truncation mutation in the
GCH gene. To clarify the mutant and normal GCH
From the *Department of Neurology, Osaka Universiy Medical
School, Suita, Osaka, and tDepartment of Medical Genetics, Nara
Medical University, Kashihara, Nara, Japan.
$Present address: Department of Neurology, Higashiosaka City
General Hospital, Nishiiwata 3-4-5, Higashiosaka 578-8588, Japan.
interaction, we performed a series of cotransfection experiments with normal GCH in different ratios with
each of three truncated GCHs, including the two previously reported by us and
Subjects and Methods
A 13-year-old girl had begun to notice, at age 11, difficulty
in walking because her right foot had begun to turn inward.
It worsened toward evening but was alleviated after sleep. It
gradually spread to the left foot, and then the proximal part
of the lower extremities began to turn inward. Neurological
examination at age 13 showed dystonic posture of both feet
and lower extremities, but there was no abnormality in the
upper extremities. She was treated with a small dose of levodopa with alleviation of dystonia. Her parents and a younger
sister were neurologically normal. There was no consanguinity. After obtaining informed consent, blood samples were
collected from the patient and parents for genetic analyses.
In addition, cDNA from the mutant gene encoding truncated GCH at amino acid (AA) 114,2 and truncated GCH at
AA 206"' were used for the present study.
Sequencing and Restriction Site Analyses of
GCH Gene
The GCH gene structure (-30 kb sequence) contains six
exons encoding a 250 AA polypeptide.' All exons and exon-
Received Jan 12, 1998, and in revised form Mar 18. Accepted for
publication Mar 19, 1998.
Address correspondence to Dr Ueno, Department of Medical Genetics, Nara Medical University, Shijo-cho 840, Kashihara, Nara
634-8521, Japan.
Copyright 0 1998 by the American Neurological Association
intron junctions were amplified by polymerase chain reaction
(PCR) and sequenced as described previously.225To confirm
a possible mutation in the present family, a primer pair
was used for a restriction site assay: E5F: 5'-GTGTCAGACTCTCAAACTGAGC-3', and E5mR 5'-ATGACGTTACTAAAGGCAGATGCAGACATA-3', carrying a substitution of A for T to create a new NdeI restriction site in the
mutant allele. To examine polymorphisms in the promoter
region, 588 base pairs upstream from the initiation codon
were PCR-amplified and sequenced. The sequences of two
Reverse Transcription-PCR of GCH mRNA
Total RNA from peripheral blood lymphocytes of the patient, her father, her mother, and normal controls were
reverse-transcribed into a first-strand cDNA, using random
hexamer primers. The primer sequences used to amplify the
cDNA containing exons 3 to 6 were F3: 5'-CATATTGGTTATCTTCCTAACA-3' and R3: 5'-GACAGACAATGCTACTGGCAGT-3'.
Quant&ation of GCH mRNA Expression
The cDNA coding for normal or mutant GCH, or coding
for p-actin, was PCR-amplified in the presence of
[a-32P]dCTP as described previously.' To ensure that quantitative data were obtained, we adjusted the number of PCR
cycles to that at which a linear relationship was detected between input RNA and the resulting final product. PCR run
consisted of 30 cycles for GCH amplification, and 15 cycles
for p-actin, for 1 minute each at 94"C, 55"C, and 72°C.
Reverse transcription-PCR (RT-PCR) products were electrophoresed on 6% polyacrylamide gels, dried, and exposed to
x-ray films and imaging plates. The radioactivities were measured with a Bio-Imaging Analyzer BAS 5000 (Fuji Film,
Tokyo, Japan). The ratios of mutant-to-normal GCH
mRNA and normal GCH-to-P-actin mRNA were calculated on the basis of the [a-32P]dCTPcontent.
sequenced. The GCH cDNA used for the transfection study
are shown schematically in Figure 1.
GCH cDNA Transfection into COS Cells
COS-7 cells were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated fetal bovine serum,
penicillin (100 U/ml), and streptomycin (100 pg/ml). The
cells were transfected with GCH-WT cDNA and mutant
GCH cDNA mixed at various ratios, using Lipofectamine
(GIBCO, Grand Island, NY), and harvested after incubation
for 48 hours.
Northern Blot and RT-PCR Analyses of
Transfected COS Cells
Total RNA from native COS-7 cells and those from transfected COS-7 cells were subjected to northern blot hybridization with [a-32P]dCTP-labeled GCH exon 1 and p-actin
cDNAs." After washing, nylon membranes (Hybond N+,
Amersham, Buckinghamshire, UK) were exposed to imaging
plates and their radioactivities measured with the image analyzer. For quantifying GCH mRNA in the cells, total RNA
treated with RQ1 RNase-free DNase (Promega, Madison,
WI) was reverse-transcribed and PCR-amplified by using F3
and Sp6 primers in the presence of [a-32P]dCTP. Sp6
primer sequence was 5'-CTAGCATTTAGGTGACACT-3'.
P-Actin mRNA was amplified as an internal control. PCR
run consisted of 23 cycles for GCH, and 17 cycles for
p-actin, for 1 minute each at 94"C, 55"C, and 72°C. The
ratios were calculated on the basis of [a-32P]dCTPcontent.
Measurement of the GCH Activity
COS-7 cells were suspended in 0.1 M Tris-HC1 buffer (pH
8.0) containing 2.5 mM EDTA and sonicated. After 15,000 g
centrifugation for 5 minutes at 4"C, GCH activity in the supernatant was assayed by release of formic acid from
Protein concentration
[8-'*C]GTP as described by others.
was determined by the method of Bradf~rd,'~
using dye reagent (Bio-Rad, Munchen, Germany) with bovine serum albumin as a standard. Activities of cotransfected cells were nor-
Fig 1. Schematic presentation of normal and mutant GTP cyPkzsmid Construction
The GCH cDNA was synthesized from lymphocyte mRNA
by RT-PCR and cloned into T vector pCRII (Invitrogen,
Carlsbad, CA). The cDNA was then excised with EcoRI digestion and ligated to the EcoRI site of expression vector
pcDNA3 (Invitrogen). The resultant plasmid contained
cDNA encoding the wild-type GCH (GCH-WT). In a similar manner, the cDNA lacking a part of exon 6 was synthesized from normal lymphocyte mRNA and cloned into
PCDNA~.'~'This mutant cDNA encoded 209 AA at the
amino terminus of the normal enzyme and was designated
GCH209. The 2 members from another HPD family that
had previously been reported by us2 had mutant transcripts
lacking exon 2 in their peripheral blood lymphocytes. The
synthesized cDNA from this mutant transcript encoded 114
AA of normal GCH, and was designated GCH114. cDNA
encoding the new GCH mutant identified in the present
family was also cloned, as described later. To eliminate any
possible PCR errors, the entire coding regions of cDNA were
366 Annals of Neurology Vol 44
No 3
September 1998
clobydrohe I (GCH) CDNA, which are cloned into expression
vector pcDN&. GCH-WT represents wild-type GCH with fill
250 amino acids (A);
GCHll4, GCH with 114 AA residues
of normal GCH; GCHl80, GCH with 180 A;and
GCH209, GCH with 209 AA. 0 = normal coding region; H
= coding region for aberrant residues; 0 = noncoding region.
exon 1
malized by the ratio of normal G C H mRNA to p-actin
mRNA, to adjust the normal enzyme level in each cotransfected sample.'*
exon 5
Generution o f Anti-GCH Antibody
The crude extract of COS-7 cells transfected with the wildtype or truncated form of G C H was prepared as described in
the enzyme assay; 40 pg of each sample was subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis on
a 15% gel for GCH114, 12% for a newly found truncated
G C H and GCH209. The proteins were transferred electophoretically to polyvinylidene fluoride membranes (Millipore, Bedford, MA) and immunoblotted with anti-GCH antibody. Immunoreactive proteins were visualized by using the
Vecta Stain ABC kit (Vector Laboratories, Burlingame, CA).
Mutunt GCH Gene
The GCH gene from the new family contained a novel
G-to-A transition at the 5' end of intron 5 in the patient (Fig 2A). Restriction site analysis showed that the
patient and her father were heterozygous for the NdeI
site, and her mother and 100 control subjects had only
the normal fragment (see Fig 2B). The sequence of the
amplified promoter regions corresponded to that reported by Witter and colleagues.''
RT-PCR analysis detected an additional 270-bp fragment in the patient and her father (Fig 3A). This small
fragment lacking exon 5 encoded 180 AA of normal
GCH on sequence analysis and was designated
GCH180 (see Fig 3B). GCH180 cDNA was synthesized from lymphocyte mRNA of the patient and cloned
into pcDNA3 for the transfection study (see Fig 1).
Mutunt und Normul GCH mRNA Expression
RT-PCR products of GCH mRNA and p-actin
mRNA were subjected to electrophoresis and autoradiography (Fig 4A). The ratio of mutant-to-normal
mRNA in the patient's lymphocytes was 2.08 ? 0.04
(mean 2 SD), significantly higher than that in the father's, 1.60 -+ 0.06 (see Fig 4B). The normal GCH/
f3-actin mRNA was 10.1 -+ 0.3 for the patient, 12.9 ?
exon 5
intron 5
C A A C g t c c g
The entire coding region of cloned GCH-WT cDNA was inserted into a plasmidexpression vector, pGEX-4T-1 (Pharmacia Biotech, Uppsala, Sweden), for synthesis of glutathione-Stransferase (GST) fusion protein in Escherichia coli. The fusion
protein was purified with bulk and RediPack GST purification
modules (Pharmacia). Every 2 weeks, two female rabbits (New
Zealand White, 12 weeks old) were injected subcutaneously
three times with purified GST-GCH fusion protein (200 pg
per injection) emulsified in 500 pl of Freund's complete adj ~ v a n t . ~The
~ , ' rabbits
were boosted intravenously with 200
pg of the fusion protein 2 weeks after the third injection. The
venous blood was obtained from the immunized rabbits 1
week after the booster injection, and the prepared anti-GCH
sera were used for immunoblotting.
Immunoblotting of Trunsfected COS Cell Extracts
intron 5
* I
cont ro I
-1 56
-1 26
Fig 2. (A) Direct sequence analysis of amplijed genomic DNA
?om a control and the patient with hereditary progressive dystonia. The patient is heterozygous for a G-to-A transition.
(B) Restriction site analysis of a mutant allelic f i a p e n t containing u G-to-A transition. The ampli$ed j a p e n t ?om
a mutant allele was digested with endonuclease NdeI
((24 TATG). Gel electrophoresis shows that the patient and
her father are heterozygousfor the mutation.
0.2 for the father, and 21.7 2 2.8 for 20 normal subjects including the patient's mother (see Fig 4C).
GCH Activity and mRNA in Transfected COS Cells
Northern blot analysis showed that native COS-7 cells
did not express GCH mRNA, whereas wild-type and
truncated G CH cDNA were transcribed in the cells
(Fig 5A). COS cells transfected with GCH-WT cDNA
showed enzyme activity, and those with GCH209
cDNA showed slight activity (-5%), but COS cells
with G C H l l 4 or GCH180 cDNA showed no activity
(see Fig 5B).
Hirano et al: G C H Mutations in H P D
fected with the expression plasmid for GCH-WT and
plasmid for GCH114, GCH180, or GCH209 in the
1:2 ratio, the enzyme activity decreased to 53%, 59%,
and 66% of control, respectively. Thus, GCHll4
caused the most profound suppression, followed by
GCH180 and GCH209, indicating that the dominant
negative effect was mutant dependent. This effect was
clearly specific, because the enzyme activity was not diminished in the COS cells cotransfected with copper/
zinc superoxide dismutase or chloramphenicol acetyltransferase cDNA (data not shown).
We have characterized the mutant GCH gene in a
new Japanese family with HPD, where a G-to-A transition at the 5’ end of intron 5 that outspliced exon
exon 4
exon 5
exon 4
5 resulted in premature termination of translation.
- 7
Whereas the full-length cDNA for normal human
Val Gln
GCH predicts 250 AA with a calculated molecular
mass of 28 kd, the GCH deduced from the mutant
transcript shares 180 AA at the amino terminus of the
normal enzyme. Both our patient with typical HPD
and her unaffected father are heterozygous for this mutation. The ratio of mutant-to-normal GCH mRNA
was higher in the patient than in her father. This clinical variability and differential transcription of the
GCH gene within this family are similar to those in
another HPD family previously reported by
explain these findings, we performed a series of cotransfection experiments.
Fig 3. (A) Agarose gel electrophoresis of reverse-transcribed and
ampl$ed mRNA coding for GTP cyclohydrohe I. The patient
GCH114 and GCH180 were expressed in COS-7
and her father present an extra band (270 bp) in adition to the
cells, as shown by RNA blotting and immunoblotting
normal band (355 bp). (B) Direct sequence analyses show that
of the cell extracts, but not by the enzyme activity.
the normal size f i a p e n t had the same sequence ac a control, but
GCH209 showed only 5% of the normal enzyme acthe mutantfiapent had aberrantjoining of a o n 4 to exon 6
tivity, suggesting that the carboxy-terminal sequence of
the last 41 AA is important for enzymatic function. By
GCH Protein Expressed in Transfected COS Cells
using coexpression with GCH-WT in COS cells, it
In immunoblots of reduced gel electrophoresis, GCH
was demonstrated that all three mutants inactivated the
protein was not detected in control COS-7 cell exnormal enzyme. Inhibition was specific, because coextracts, whereas in transfected COS cells with
pression of GCH with unrelated proteins had no effect
GCH-WT cDNA, protein was detected with an apparon enzyme activity. That GCH114 was most effective
ent molecular mass (28 kd) for the monomeric form of
in reducing the enzyme activity and then GCH180
the normal enzyme. GCH114 (13 kd), GCH180 (21
and GCH209 in this order indicates that the dominant
kd), and GCH209 (24 kd) were also expressed and denegative effect is mutant dependent.
tected by the antibody. Coexpression of GCH-WT with
This is in agreement with the conclusion of the
each of the three mutants is also shown in Figure 6.
study of the scavenger receptor by Dejager and colleagues18 showing a greater effect of inhibition by a
Inhibition of Normal Enzyme Activity by
larger truncation.
Mutant GCH
Plausible mechanisms for dominant negative effects
GCH activity was determined with COS-7 cells cocan be deduced from the multimeric structure of
rransfected with GCH-WT cDNA and mutant GCH
GCH; mutant subunits capable of interacting with
cDNA in various ratios. Their transcripts were deterwild-type ones may be inhibitory. In fact, native GCH
mined by RT-PCR combined with the image analyzer.
is a decamer formed by fivefold symmetrical arrangeAs shown in Figure 7, coexpression of GCH-WT with
ment of the d i m e r ~ . ” ,Although
the precise mechaeach mutant GCH resulted in a remarkable decrease of
nism by which inhibition of the enzyme function octhe enzyme activity. When COS cells were cotranscurs is not certain, it is possible that the mutant may
Annals of Neurology Vol 44
No 3
September 1998
Fig 4. (A) Autoradiograph of reverse transcription-polymerase chain reaction (RT-PCR)
of mRNA, codingfor normal (355 bp) and
mutant (270 bp) GTP cyclohydrohe I
(GCH) and p-actin (218 bp). Pt = patient;
Fa = father. (B) The expressed ratio of
mutant-to-normal GCH mRNA was higher
in the patient than her asymptomaticfather.
Quantification was performed with the normal and mutant j a p e n t s ampl$ed by five
separate RT-PCR amplifications. 9 < 0.05,
repeated analysis of variance. (C) Ratios of
normal GCH-to-P-actin mRNA for the patient, her father, and controls. 9 < 0.05,
repeated analysis of variance.
I 2.0
p a c tin
Pt Fa
Pt Fa
- 18s
' *
Fig 5. (A) Northern blot analysis shows that GTP cyclohydrolase I (GCH) mRNA was not detected in nontransfected COS-7 cells
but was present in the cells transfected with wild-type GCH (GCH-WT) cDNA and mutant GCH cDNAs. GCH-WT represents
wild-type GCH with j W 250 amino acid (AA); GCH114, GCH with 114 AA residues of normal GCH; GCH180, GCH with
180 AA; and GCH209, GCH with 209 AA. (B) The activity of COS-7 cells transfected with GCH-WT cDNA was 0.447 2
0.03 nmol/hr/mg of protein (mean 2 SO). The native COS-7 cells and COS-7 cells with GCHll4, or GCHl8O expression,
showed no enzyme activity, whereas the activity in COS-7 cells with GCH209 expression was 0.022 2 0.001 nmol/hr/mg ofprotein. 9 < 0.05, repeated analysis of variance. Assays were done in quadruplicate.
exert its inhibitory effect by forming heteromers with
the native enzyme, leading to formation of nonfunctioning multimers, to aberrant processing, or to premature degradation of the native enzyme. An alternate
possibility is that increased expression of the mutanr
subunits may reduce synthesis of the normal enzyme.
The results of the in vitro experimentation may in part
explain variation of the clinical phenotype within the
same family members. We have previously reported that,
in the HPD family associated with GCHll4 mutant, the
mutant/normd GCH mRNA ratio was 0.28 for the affected carrier, and 0.083 for the unaffected carrier.2 Figure
7 shows that this difference corresponded to an approximately 25% decrease in enzyme activity, which may be
sufficient enough to produce phenotypic differences in
these two carriers. In the family with GCH180, the difference in the ratio of the affected (2.08) and unaffected
carriers (1.60) may produce about 5% difference in the
Hirano et al: GCH Mutations in HPD
- 30
- 30
- 30
- 20
- 20
- 20
- 14
Fig 6. (A) Immunoblotting demonstrates that GTP cyclohydrolase I (GCH) subunit was expressed in the COS-7 cells transfected
with the expression vector f i r wild-type GCH (GCH-Wr) and GCH truncated at 114 (GCH114) but was not detected in nontransfected cells. Coexpressed cells possessed both GCH- WT and GCHll4 subunits. Molecular mass standards are indicated at right
(kd). (B) The expression of GCH truncated at 180 (GCH180). (C) The expression of GCH truncated at 209 (GCH209).
m so/
2.0 3.0
4.0 5.0
mutanthormal GCH mRNA
mutantlnormal GCH mRNA
3.0 4.0
mutanthormal GCH mRNA
Fig Z GTP cyclohydrobe I (GCH) activity of COS-7 cells coexpressed with wild-type GCH (GCH-WT) and a mutant GCH.
GCH-WT represents wild-type GCH with fill 250 amino acids (A)
GCH with 114 AA residues of normal GCH
(A); GCH180, GCH with 180 AA (B); and GCH209, GCH with 209 AA (C). For each set, 100% GCH-WT mRNA is expected to have 100% activity, and other points are expressed as percentages relative to this control. The dashed line indicates the
GCH activity when the wild-type and truncated forms of GCH protein presumably acted independently. Autoradiographs (insets)
show reverse-transcribedpolymerase chain reaction of normal (upper band) and mutant (lower band) GCH mRNA.
enzyme activity. Whether this small change can lead to
differences in clinical phenotype is unknown; however,
the possibility exists that such a slight decrease in enzyme
activity may increase the risk for reaching the threshold to
cause dystonia. In contrast, in the new mutation reported
in the present study, the absolute level of normal enzyme
expression might be more important. In fact, expression
of normal GCH mRNA, and presumably the enzyme
protein level, is lower in the patient than an unaffected
carrier (see Fig 4).
One important question is whether a naturally occurring truncation leads to severe functional defects.
We have shown that GCH209 is a dominant negative
Annals of Neurology
Vol 44
No 3
September 1998
mutation; however, it cannot inactivate normal enzyme
activity to a level below about 65% of control. The
absolute predominance of this form over the native
form did not exist in normal tissues.' From these facts,
we concluded that GCH209 might be clinically innocent, but possibly taking part in posttranscriptional
regulation of the G C H gene expression, as the transcription factor E3 gene does.21
Taken together, we have shown that truncated
GCH, by which the normal G C H function is inhibited, may be responsible for the clinical manifestation
of some patients with HPD. However, the following
several questions and problems still require further at-
tention: (1) Why do individuals with the same mutation show different mutant/normal mRNA ratios? Although promoter sequence variations are known to
alter the particular allelic expression in some other
genes,22,23 we failed to detect any variation in the
GCH promoter. Clarification of the mechanism for
the observed differential transcription awaits further investigation. (2) Is the mutanthormal mRNA ratio
consistent within an individual over time? The ratios in
our previous cases235remained constant for 4 months,
but we do not yet have a longer period of observation.
( 3 ) Why is there a sex difference (malelfemale ratio,
1:4) in HPD?” The mechanism for the female predominance may be explained if sex hormone-responsive
elements are identified in the GCH gene. (4) Mutations
have been reported in the first or the second codon of
the G C H gene.’,’ These mutations, due to frameshifting, would produce subunit peptides incapable of interacting with the normal subunit. In these mutations,
other mechanisms may explain the clinical manifestation. ( 5 ) GCH activities in lymphocytes were too low to
be measured in the present study. As Ichinose and coworkers’ have done, peripheral blood mononuclear cells
can be stimulated with phytohemagglutinin to induce
the enzyme. However, GCH activities of artificially
stimulated lymphocytes may not correspond to GCH
mRNA levels of the native lymphocytes.
In conclusion, the dominant negative effect of mutant
GCH on the normal enzyme may be one of the molecular mechanisms that determine the diversity of clinical
phenotypic expression of HPD, and is one of the most
interesting aspects of dominantly inheritable diseases.24325
This study was supported by a Nakajima Memorial Research Grant
(S.U.) and a Nara Medical University Research Grant, and by a
Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (S.U.).
We are grateful to Dr Kurumatani for his assistance in the statistical
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Hirano et al: GCH Mutations in HPD
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effect, progressive, dopa, mutation, gtp, dominantly, responsive, dystonic, hereditary, negativa, cyclohydrolase
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