Mutation in intron 5 of GTP cyclohydrolase 1
gene causes dopa-responsive dystonia
(Segawa syndrome) in a Brazilian family
C.P. Souza1, E.R. Valadares2, A.L.C. Trindade1, V.L. Rocha2, L.R. Oliveira3
and A.L.B. Godard1
1
Departamento de Biologia Geral, Laboratório de Genética Animal e Humana,
Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais,
Belo Horizonte, MG, Brasil
2
Departamento de Propedêutica Complementar, Faculdade de Medicina,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil
3
Departamento de Pediatria, Faculdade de Medicina,
Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil
Corresponding author: A.L.B. Godard
E-mail: brunialti@icb.ufmg.br
Genet. Mol. Res. 7 (3): 687-694 (2008)
Received April 14, 2008
Accepted June 23, 2008
Published August 5, 2008
ABSTRACT. Dopa-responsive dystonia (DRD), also known as Segawa
syndrome or hereditary progressive dystonia with diurnal luctuation, is
clinically characterized by the occurrence of simultaneous or late Parkinsonism and by an excellent response to treatment with low doses of
L-dopa. Diagnosis of DRD is essentially clinical. It is based on clinical
history and the response to treatment with low doses of L-dopa. However, due to the low penetrance of the disease, asymptomatic carriers may
exist. In these cases, mutational analysis of the GCH1 gene is an alternative to diagnose DRD. In the present study, we investigated a large
DRD-carrier family in an attempt to identify the disease-causing mutation. The proband, a young woman diagnosed at the age of 13 years, is
the daughter of a healthy non-consanguineous couple with history of several cases, on the maternal side of the family, of tip-toeing, disturbance of
gait, Parkinsonism, rigidity and cramps in the lower limbs. Using single
Genetics and Molecular Research 7 (3): 687-694 (2008)
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C.P. Souza et al.
strand conformational polymorphism and DNA sequencing techniques to
analyze DNA extracted from blood samples, we identiied a mutation in
the GCH1 gene, IVS5+3insT, which would preclude the formation of the
active enzyme due to the formation of truncated peptides.
Key words: Dopa-responsive dystonia; Segawa syndrome;
GCH1 gene; GTP cyclohydrolase; Negative dominant effect;
Hereditary progressive dystonia with diurnal luctuation
InTROduCTIOn
Dopa-responsive dystonia (DRD), also known as Segawa syndrome or hereditary
progressive dystonia with diurnal luctuation, was described for the irst time by Segawa
and colleagues in 1971. The disease is clinically characterized by the occurrence of simultaneous or late Parkinsonism and by an excellent response to treatment with low doses of
L-dopa. In most cases, clinical manifestations start in childhood with dystonia, more frequently, in the lower limbs (Van Hove et al., 2006). In these cases, DRD phenotype may be
similar to atypical cerebral palsy. Thus, about 24% of the patients with DRD are incorrectly
diagnosed with cerebral palsy (Jan, 2004). In adults, Parkinsonism can be the main or the
only symptom. Segawa syndrome is a rare disorder and occurs at a frequency of 1 in 2 million (Nygaard, 1993). However, this rate is probably underestimated due to, at least, two
reasons: the irst one is that the disease is not easily recognized by physicians; the second
reason lays in the fact that many signs and symptoms are not severe enough for patients to
seek clinical advice (Bianca and Bianca, 2006).
DRD can be inherited as an autosomal dominant or in a recessive manner, depending on the mutated gene (locus heterogeneity). The recessive condition is caused by a
mutation in the tyrosine hydroxylase gene, which is a mono-oxygenase that catalyzes the
conversion of L-tyrosine into L-dopa (De Lonlay et al., 2000). In this study we emphasized the dominant condition caused, mainly, by mutations in the guanosine triphosphate
cyclohydrolase 1 gene (GCH1), located on chromosome 14q22.1-q22.2 (Nygaard et al.,
1993). The disease, inherited as an autosomal dominant, exhibits reduced penetrance,
highly variable expressivity and, it is estimated that women are two to four times more
affected than men (Ichinose et al., 1994). The GCH1 gene is constituted by 6 exons; it
occupies approximately 30 kb in the genomic DNA and, at least six differential splicing
variants differing on the 3’ end have been described (Togari et al., 1992; Golderer et al.,
2001; Hwu et al., 2003). However, only the full length transcript exhibits enzymatic activity (Gütlich et al., 1994). This transcript codes GTP cyclohydrolase I, an enzyme involved
in the regulation of tetrahydrobiopterin (BH4) synthesis, an essential co-factor for hydroxylases that converts phenylalanine, tryptophan, and tyrosine into tyrosine, serotonin
and L-dopa, respectively (Müller et al., 2002).
Diagnosis of DRD is essentially clinical, based on the clinical history of the patient and the response to treatment with low doses of L-dopa. However, due to the low
penetrance of the disease, asymptomatic carriers may exist. In these cases, the mutational
analysis of the GCH1 gene is an alternative to detect asymptomatic and oligosymptomatic
carriers. Alterations in the gene sequence have been found, through single strand conforGenetics and Molecular Research 7 (3): 687-694 (2008)
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mation polymorphism (SSCP) analyses, in about 50 to 60% of clinically identiied cases
(Hagenah et al., 2005). Recently, a heterozygous deletion, not detectable through conventional sequencing, was identiied by Southern blot analysis (Furukawa et al., 2000).
The objective of this study was to identify the mutation which causes DRD in individuals of an affected family.
MATERIAL And METhOdS
Patients
Fifty-ive individuals from the same DRD Brazilian family were studied. The proband
was a young woman diagnosed when she was 13 years old, who was the daughter of a healthy
non-consanguineous couple with history of several cases of tip-toeing, disturbance of gait,
Parkinsonism, rigidity and cramps in the lower limbs on the maternal side of the family. It is
known that her pregnancy and delivery were free of incidences. Her symptomatology started
at around 1 year old with dystonia in the left foot, progressively evolving to dystonia of the
lower limbs. The affected patient was submitted to a posterior left tibial tenotomy on the age
of 10 years. At 13 years, she could not walk more than one block, and she complained about
an almost constant lack of balance and decrease in spasticity when at rest. The patient had
previously shown unaltered results in electromyography, brain tomography and encephalic
magnetic resonance. She had been incorrectly diagnosed by several neurologists with spastic
diplegia, and physical therapy was the only treatment indicated for her. In the objective examination, she showed dystonia of both lower limbs, mild axial dystonia, latfeet, exaggerated tendon relexes and Babinski sign. Total remission of symptoms was achieved when the
treatment with L-dopa was initiated (30 mg twice a day, orally). The diagnosis for DRD was
then established. The same treatment was indicated for all affected and symptomatic relatives.
Blood samples of all ifty-ive individuals were collected for DNA extraction.
The participants were fully informed and participated voluntarily. The project was approved by the Research Ethics Committee of the Universidade Federal de Minas Gerais under
the number CAAE - 0428.0.203.000-05, February 15, 2006.
Molecular analyses
Genomic DNA was extracted from 10 mL peripheral blood according to a standard
protocol: lysis buffer (10 mM Tris-HCl, pH 8.0, 2 mM EDTA, pH 8.2, and 10% SDS) and
proteinase K (20 mg/mL). Then, the pellet was washed with 70% ethanol and dissolved in TE
(Miller et al., 1988). The DNA was quantiied by spectrophotometry at 260 nM.
The exons of the GCH1 gene, including the splicing sites, were amplified from
genomic DNA using polymerase chain reaction with previously described primers (Ichinose et al., 1995). For identification of the mutated fragment, the SSCP analysis was
applied to the amplification products of the family members and the individual control.
The fragment that exhibited a different migration pattern in the gel when the two groups
were analyzed was cloned into the pCRII-TOPO plasmid using the TOPO TA Cloning Kit, as recommended by manufacturer instructions (Invitrogen™ Life Technologies,
Carlsbad, CA, USA) and, then, both alleles were sequenced in order to identify the mutaGenetics and Molecular Research 7 (3): 687-694 (2008)
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tion causing the disease. Sequencing was done with the same primers used in the initial
amplification, in a MegaBACE 1000 system (GE Healthcare) using the DYEnamic ET
Terminator kit according to manufacturer instructions. Both strands of the DNA samples
were sequenced. The sequences obtained were first converted into the ab1file format, using the Chromas software (www.technelysium.com.au/chromas_lite.html), and then analyzed with the CodonCode Aligner program (www.codoncode.com/aligner). The fragment
containing exon 1 was directly sequenced since, because of its size (502 bp), it was not
adequate for the SSCP technique.
RESuLTS
The characteristic of an autosomal dominant inheritance can be observed in Figure 1, which shows the pedigree of the family. Except for exon 5, the migration pattern of
the fragments on the SSCP gel did not exhibit differences between the individuals studied
and the control sample. Figure 2 shows that, individuals VI-3, IV-35, III-12, and V-16
presented the same migration pattern as the individual control, thus, they, probably, do
not have a mutation. The remaining individuals showed an extra band, and are, therefore,
carriers of the mutation.
Figure 1. Pedigree of a Brazilian family with dopa-responsive dystonia. Nineteen of the ifty-ive individuals
analyzed are mutation carriers.
Figure 2. Single strand conformation polymorphism gel showing the fragment containing exon 5 of the GCH1 gene.
The arrow indicates the band that differs from the banding pattern observed in the control sample (C). Individuals who
have the mutation show two bands. The numeration is in accordance with the pedigree in Figure 1.
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Direct sequencing of the fragment containing exon 1 did not indicate any mutation in
DNAs analyzed. On the other hand, sequencing of the fragment containing exon 5 showed a
heterozygous splice site mutation, the insertion of a thymine next to the splice site of intron 5.
Figure 3 shows the chromatogram with the sequencing of the cloned fragment of exon 5 from
the individual with the mutation.
Figure 3. Chromatograms of the sequencing of the exon 5 of the GCH1 gene from a mutated individual that was
cloned (IV-23). Comparing with the GCH1 dataset (www.ensembl.org), it is observed that the mutated allele shows
one more peak that represents an insertion of a thymine.
The mutation IVS5+3insT in the GCH1 gene was identiied in 19 individuals, 15
women and 4 men, of the 55 individuals studied. Nine women were symptomatic (60% of the
detected women), and only 1 man (25% of the detected men).
dISCuSSIOn
More than 85 mutations in the GCH1 gene have been described so far (http://www.
bh4.org/BH4_databases_biomdb2.asp) and most of them reside in the coding region of the
gene. Abnormalities have been reported in all 6 exons of the gene and in the intron splicing
sites (Nishiyama et al., 2000). In this study, the mutation in the intron 5 splicing site of the
GCH1 gene, IVS5+3insT, was identiied in 19 individuals of a Brazilian family, as the one
causing DRD. This mutation has already been described in patients with DRD (Kaindl et al.,
2005). The insertion probably precludes the intron 5 remotion and causes the introduction of a
premature stop codon resulting in a truncated peptide without exon 6.
The enzyme guanosine triphosphate cyclohydrolase 1 is a protein composed of ten
identical 250 amino acid monomers, coded by the GCH1 gene. The homodecamer is formed
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by ive dimers organized in a ring structure that looks like two pentamers aligned face to face
(Nar et al., 1995). Figure 4 shows a diagram with the relationship between the exons of the
GCH1 gene and the structural part of the peptide for which they code. The study of Swick and
Kapatos, published in 2006, showed that the dimers associate in a decamer through the interaction between b1 of one of the monomers with b4 of another monomer from an adjacent dimer.
In addition, they have also concluded from their experiments that h6 would also stabilize the
decamer. Based on Figure 4 it is possible to infer that the truncated peptide would be deicient
in h6 α-helices and b3 and b4 β-sheets that correspond to exon 6. This deiciency would prevent
the formation of the decamer through the dimer interaction, since, as shown previously, this
interaction takes place between segments b1 and b4 of the monomer. Biochemical analyses
showed that only the GCH1 decamer exhibits enzymatic activity (Yim and Brown, 1976); this
fact is explained by structural studies that indicate the existence of 10 active sites formed on the
interface of three separate monomers, located in adjacent dimers (Nar et al., 1995). Thus, it can
be suggested that the product coded by the mutated gene would not show enzymatic activity.
Another fact that supports this hypothesis is that the truncated peptide would not code one of
the amino acids, Cys 212, which is part of the active site (Auerbach et al., 2000).
Figure 4. Diagram showing the correspondence between exons and structural parts of the GTPCH1 peptide.
Each monomer folds into an N-terminal α-helix (h1) and two anti-parallel helices (h2, h3), that are separated by
a compressed C-terminal domain, composed of four anti-parallel β-sheets (b1-b4) divided by two anti-parallel
helices (h4, h5) that end in a C-terminal helix (h6) (Nar et al., 1995).
Many inborn errors of the metabolism are recessive because half of the enzymatic activity is enough to produce or metabolize a certain substance (Suzuki et al., 1999). In DRD this
haplosuficiency is not observed and it is estimated that the activity of GTP cyclohydrolase in
the brain is reduced to 20% compared to normal individuals (Suzuki et al., 1999), generating a
dominant inherited disease. One hypothesis that was believed initially to explain the observed
fact was the possibility that the mutant and normal peptides interacted forming a non-functional
heterodecamer, i.e., the mutant peptide would exert a negative dominant effect over the wild
type (Hirano and Ueno, 1999). These authors were able to demonstrate this effect for some
mutations in co-transfection experiments with wild-type and mutated GCH1 cDNAs. However,
some mutations have been described, which code peptides apparently unable to interact with
wild-type peptides (Suzuki et al., 1999). Experiments done by Hwu and colleagues, in 2003,
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have indicated that the presence of GCH1 transcript isoforms, type II mRNA - which does not
show enzymatic activity - contributes to the regulation of the gene by reducing the basal activity
of the protein through a dominant negative effect. Thus, another possible hypothesis would be that
in DRD patients, because of the decrease in the amount of a functional protein (type I), there is
an increase in the type II mRNA/type I mRNA ratio resulting in a greater chance of assembling
non-functional heterodecamers (Hirano et al., 1997). Since the peptide coded by the mutated allele
described here would, probably, be unable to form heterodecamers due to the reasons mentioned
above, the second hypothesis would be more adequate to explain the haploinsuficiency of the
normal allele in this case.
In the group of 19 individuals with the mutation, women were symptomatic in 60% of
cases (9 in 15) and men in 25% (1 in 4).
In summary, we have identiied the mutation at the GCH1 gene, IVS5+3insT, as the
cause of DRD in a Brazilian family.
ACKnOwLEdGMEnTS
We thank all members of the studied family that willingly participated in this research.
Research supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Brazil.
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