Novel Mutations in the Guanosine Triphosphate Cyclohydrolase 1 Gene Associated with DYT5 Dystonia

ORIGINAL CONTRIBUTION Novel Mutations in the Guanosine Triphosphate Cyclohydrolase 1 Gene Associated With DYT5 Dystonia

Etsuro Ohta; Manabu Funayama, PhD; Hiroshi Ichinose, PhD; Itaru Toyoshima, MD; Fumi Urano; Mitsuhiro Matsuo, MD; Nishida Tomoko, MD; Konishi Yukihiko, MD; Syuji Yoshino, MD; Hiroyuki Yokoyama, MD; Hideki Shimazu, MD; Koji Maeda, MD; Kazuko Hasegawa, MD; Fumiya Obata, PhD

Objectives: To better understand the relationship be- helix structure of the enzyme. In the third patient, we tween mutation of the guanosine triphosphate cyclohy- found a new mutation (a 15–base pair nucleotide dele- drolase I (GCH1) gene and the etiology of DYT5 dysto- tion) in exon 5 that may cause a frameshift involving the nia and to accumulate data on the mutation in the Japanese active site. In the fourth patient, we detected a known population for genetic diagnosis of the disease. nucleotide GϾA substitution in the splice site of intron Setting: Japanese population. 5, which has been reported to produce exon 5–skipped messenger RNA. The concentrations of both neopterin Patients: Eight Japanese patients with suspected DYT5 and biopterin in the cerebrospinal fluid of the third and dystonia were analyzed. fourth patients were markedly lower than the normal range, indicating that the GCH1 enzyme was function- Intervention: Direct genomic sequencing of 6 exons of GCH1 was performed. ally abnormal in these mutations. Gene dosage analysis showed that the fifth patient had a deletion of both exon Main Outcome Measures: For patients who did not 3 and exon 4, whereas the sixth patient had a deletion of exhibit any abnormality in the sequence analysis, the pos- exon 3. sibility of exon deletions was examined. In cases for which cerebrospinal fluid was available, the concentrations of Conclusions: We found several novel, as well as known, neopterin and biopterin were measured as an index of GCH1 mutations in Japanese patients with DYT5 dysto- GCH1 enzyme activity. nia. In some of them, the GCH1 enzyme activity was proved to be impaired. Results: In 2 patients, we found a new T106I mutation in exon 1 of GCH1, a position involved in the helix-turn- Arch Neurol. 2006;63:1605-1610

YT5 DYSTONIA (SEGAWA The human GCH1 gene is composed of disease) is an autosomal 6 exons spanning approximately 30 kilo- dominant hereditary pro- bases.9 Todate,varioustypesofmutationhave gressive dystonia with been found throughout the 6 exons, as well marked diurnal fluctua- as introns, of the GCH1 gene, including mis- tion,D characterized by bilateral foot dysto- sense and nonsense mutations, large and nia that becomes apparent in childhood or small deletions and insertions, and splice site adolescence.1 Molecular genetic studies have mutations3,4,10-16 (Figure 1). The reason why revealed that DYT5 dystonia is caused by suchavarietyofmutationsoccurintheGCH1 mutations of the guanosine triphosphate cy- gene remains unknown. Patients with DYT5 clohydrolase I (GCH1) gene located in dystonia in each pedigree, but not in differ- 14q22.1-22.2.2-4 This enzyme catalyzes the ent pedigrees, have identical mutations. It is rate-limiting step of tetrahydrobiopterin bio- not known how the single GCH1 gene is as- synthesis.5 Tetrahydrobiopterin is a cofac- sociated with 2 distinct diseases, DYT5 dys- tor for tyrosine hydroxylase, which is in- toniaandhyperphenylalaninemia.Inourpre- volved in the production of dopamine.6 It viousshortreport,wedescribedanovelT106I remains to be clarified, however, how a low mutation found in exon 1 of the GCH1 gene dopamine level results in the onset of this in patients with DYT5 dystonia.17 In the disease. Tetrahydrobiopterin is also a co- present article, we provide further details of factor for phenylalanine hydroxylase, and this mutation. In addition, analysis of other defective activity of this enzyme causes hy- patients revealed a novel small deletion and Author Affiliations are listed at perphenylalaninemia, which is clinically dis- a new type of exon deletion, as well as the the end of this article. tinct from DYT5 dystonia.7,8 known splice site mutation.

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 M1X R88P E2fs R88Q89del Q182X A8X H210X Q89X R184H P23L M211I Q89 – P95del R184H Q48X M211V G90V R184C P49fs M211fs L91R L185R E56X M213V T94K T186K E61X M213T W96X I189X E65X R216X M102K V191I P69L V218fs M102R S196A N70fs M221T M102fs P199A L71Q S223R Q103P P199L A74V K224R ins106F G201E Y75X L117X K224X G201R S76X T106I D134V V226A V202I S77–L82del G108D I135K I171X F234S G203R L79P G108V M137R S176T R241W V204I G83A Q110fs F138fs R178S E242fs V205E Q87P Q110X C141R H153P R178G E243fs V205G R88G S114X C141W N159X Q180X R249S –22C>T R88W D115N H144P L163R Q180R V206fs X251R –39C>T/ –132C>T

Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 IVS1–1G>A IVS2+1G>C IVS3+1G>T IVS4+1G>C IVS5+1G>A IVS1–2A>G IVS2 – 2A>G IVS3–2G>A IVS4+1G>A IVS5+5G>A Deletion Exon 1 IVS5+3insT Deletion Exons 1-3 Deletion Exons 1-4 Deletion Exons 1-6 Deletion Exon 3 Deletion Exons 3-4

Figure 1. Cumulative mutations of the guanosine triphosphate cyclohydrolase I (GCH1) gene detected in patients with DYT5 dystonia or hyperphenylalaninemia. The Figure has been modified from that of Blau and Thony (http://www.bh4.org) (2003) by including additional mutations. The mutations found in this study are indicated by arrows. Mutations associated with hyperphenylalaninemia are underlined. fs indicates frameshift; del, deletion; and x, stop codon.

Table 1. Clinical Characteristics and GCH1 Mutations of 8 Patients Analyzed in This Study

Patient/ Family History Levodopa/DCI Sex/Age, y GCH1 Mutation Site of Onset Clinical Signs of Dystonia Effective Dose, mg/d P1/F/8 T106I Leg Foot dystonia Not known 100 P2/F/30 T106I Arm Writer’s cramp; foot dystonia Yes 200 P3/F/7 V206fs Arm, leg, neck Cervix rotation Yes Data unavailable P4/F/4 IVS5 ϩ 1GϾA Unknown Foot dystonia Not known 400-600 P5/F/8 Exons 3-4 deletion Arm Dystonia of lower and upper limbs Yes 300 P6/M/8 Exon 3 deletion Leg Foot dystonia Not known Data unavailable P7/F/20 Not detected Trunk Anteversion posture Not known Data unavailable P8/F/18 Not detected Leg Tremor Not known 500

METHODS HAPLOTYPE ANALYSIS Five microsatellite loci (D14S288, D14S978, D14S991, D14S1057, POLYMERASE CHAIN REACTION and D14S980) around the GCH1 gene of patients P1 and P2 were DIRECT SEQUENCING amplified by PCR using fluorescence-labeled primers for each locus. Allele frequencies in the Japanese population at each locus Genomic DNA was isolated from peripheral blood leukocytes were obtained by genotyping 54 Japanese volunteers. of 7 female patients (P1, P2, P3, P4, P5, P7, and P8) and 1 male patient (P6) with clinically suspected DYT5 dystonia (Table 1) ANALYSIS OF 15–BASE PAIR DELETION and 100 healthy control individuals, with the informed con- IN THE NORMAL POPULATION sent of the donors. The 6 exons of the GCH1 gene were amplified by polymerase chain reaction (PCR) using the primers The exon 5 genomic sequence around a 15–base pair (bp) de- reported by Ichinose et al3 and subjected to sequence analysis. letion in 100 normal individuals was amplified using fluores-

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 cence-labeled primers, and the PCR products were analyzed by GeneScan. T T C T T C A G C A A G G G C MEASUREMENT OF NEOPTERIN P1 C317C/T AND BIOPTERIN CONCENTRATIONS (Thr106Ile)

Concentrations of neopterin and biopterin in cerebrospinal fluid T T C T T C A G C A A G G G C (CSF) were measured by high-performance liquid chromatog- P2 18 C317C/T raphy (HPLC) as described previously. In brief, the CSF was (Thr106Ile) oxidized in iodine solution. The oxidized samples were analyzed by HPLC using an organized delivery system reverse- phase column (GL Sciences). T T C T T C A G C A A G G G C C317C GENE DOSAGE ANALYSIS Control

Six exons of the GCH1 gene were subjected to real-time PCR analysis using SYBR Green PCR Master Mix (Applied Biosystems, Fos- Mutant GCCATGCAGTTCTTCATCAAGGGCTACCAGGAGACCATC ter City, Calif) and a PRISM 7700 Sequence Detection System A M Q F F I K G Y Q E T I Human GCCATGCAGTTCTTCACCAAGGGCTACCAGGAGACCATC (Applied Biosystems). The Ct value for each exon was normal- A M Q F F T K G Y Q E T I ized using those for the beta-globin gene. A ratio between 0.8 and Rat A M Q F F T K G Y Q E T I 1.2 was considered normal, and a ratio between 0.4 and 0.6 was Mouse A M Q F F T K G Y Q E T I considered to represent a heterozygous deletion. Chicken A M Q F F T K G Y Q E T I Xenopus A M Q F F T K G Y Q E T I RESULTS

Figure 2. T106I mutation of exon 1 found in patients P1 and P2. The ANALYSIS OF PATIENTS nucleotide and amino acid change, as well as those of other species, are WITH A MISSENSE MUTATION shown in the lower part of the Figure. GenBank accession numbers are as follows: human, Z29434; rat, M58364; mouse, L09737; chicken, Z49267; and Xenopus, BC075602. Eight patients (P1, P2, P3, P4, P5, P6, P7, and P8) who had been suspected to have DYT5 dystonia from their levodopa responsiveness and clinical characteristics (Table 1) were analyzed. The 6 exons of the GCH1 gene of these pa- Table 2. Microsatellite Haplotypes Around GCH1 tients were amplified by PCR and subjected to direct se- of the Patients (P1 and P2) With the T106I Mutation quence analysis. In 2 patients (P1and P2), we found a novel and identical heterozygous mutation. These patients had P1 P2 Ͼ Microsatellite Frequency of a C317 T nucleotide mutation in exon 1, leading to an Loci Shared Allele amino acid substitution of T106I (Figure 1 and Figure 2). D14S288 207† 205 207† 211 0.176 To our knowledge, this mutation has not been reported pre- D14S978 254† 256 254† 260 0.546 viously in Japanese or other populations and was not de- D14S991 171† 159 171† 167 0.065 tected in the 100 normal controls, indicating that it does GCH1* D14S1057 161† 161 161† 167 0.324 not represent a normal polymorphism. Since the 2 pa- D14S980 172† 160 172† 174 0.102 tients (P1 and P2) were not aware of any blood relationship with each other but lived close together, we per- *The position of GCH1 is shown. †Indicates the alleles shared by the 2 patients, whose frequencies are formed haplotype analysis using 5 microsatellite markers shown in the right-hand column. mapped around the GCH1 gene. We discovered that the 2 patients shared 1 of the 2 alleles of each locus (Table 2). The probability of sharing all the 5 alleles is calculated to be 2.1ϫ10−4 (1 in about 5000) according to the frequen- GeneScan, indicating that the mutation does not repre- cies in the Japanese population, suggesting that they origi- sent a normal polymorphism. nated from a common founder. A CSF sample from this patient was available, and the concentrations of neopterin and biopterin, the meta- ANALYSIS OF A PATIENT bolic byproducts of tetrahydrobiopterin biosynthesis, were WITH A SMALL DELETION measured by HPLC to assess the enzyme activity of GCH1. The concentrations of neopterin (8.5 pmol/mL) and bi- In 1 patient (P3), we found a novel heterozygous 15-bp opterin (8.2 pmol/mL) were found to be markedly lower deletion that, to our knowledge, has not been reported than the normal range, indicating that this mutation leads previously in Japanese or other populations. The dele- to functional impairment of the GCH1 enzyme (Table 3). tion, starting from T618 in exon 5, results in amino acid– sequence frameshifts starting from V206 (Figure 1 and ANALYSIS OF A PATIENT Figure 3). All of the possible 3 frameshifts give rise to WITH A SPLICE SITE MUTATION downstream termination codons and would disrupt the active site of the GCH1 enzyme.19,20 This deletion was not In 1 patient (P4), we detected a GϾA nucleotide substi- detected in the 100 normal controls when analyzed by tution in the splice site of intron 5 (Figure 1). This mu-

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P3 (Va1206fs)

G G G T A G T G G T T G A A G C A A C G T A A G T C T G C A

Control

618 Mutant 5´-GGGTAGTGGT ctgca-3´

Normal 5´-GGGTAGTGGTTGAAGCAACgtaagtctgca-3´

200 206 250

Wild Type AGVGVVVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS

Frameshift 1 AGVGVVVCICL∗

Frameshift 2 AGVGVVTHVYGNARCTENEQQNCDQHNVGCVPGGSKDSGRVPDSH∗

Frameshift 3 AGVGTHVYGNARCTENEQQNCDQHNVGCVPGGSKDSGRVPDSH∗

Figure 3. The 15–base pair deletion of exon 5 found in patient P3. The nucleotide sequence around the break point of the deletion (top and middle) is shown. The 3 different possible amino acid–sequence frameshifts from V206 (frameshift 1, frameshift 2, and frameshift 3) are shown (bottom). All of the 3 frameshifts cause a downstream termination codon (indicated by an asterisk).

tation had been reported to produce messenger RNA with- A CSF sample from patient P5 was available and was out exon 5 and cause impaired activity of recombinant found to contain concentrations of neopterin (8.6 pmol/ enzymes, although the enzyme activity in the affected pa- mL) and biopterin (15.7 pmol/mL) that were both lower tient had not been analyzed.11 In the present study, we than the normal range, although to a lesser extent in the found that the concentrations of neopterin and biop- latter case (Table 3). terin in the CSF of the patient were 3.9 pmol/mL and 1.3 pmol/mL, respectively (Table 3). These concentrations were strikingly lower than the normal range, indicating COMMENT that this mutation markedly impairs the function of the GCH1 enzyme. In our previous short report, we described a novel T106I mutation in the GCH1 gene in 2 patients with ANALYSIS OF PATIENTS DYT5 dystonia.17 In the present article, we have pro- WITH EXON DELETIONS vided more detailed results and discussion related to this mutation. T106 is conserved in human, mouse, rat, In 4 patients (P5, P6, P7, and P8), we did not detect any chicken, and Xenopus and is located at the turning posi- mutation by sequence analysis of the 6 exons. There- tion of the helix-turn-helix structure of the guanosine fore, gene dosage analysis was performed by real-time triphosphate cyclohydrolase I molecule.9,19,20 Because PCR. It was found that in 1 patient (P5) the gene dos- the G90V mutation located at the same helix-turn-helix age values for exons 3 and 4 were 0.42 and 0.60, structure has been reported to cause a dominant nega- respectively, whereas in another patient (P6), the value tive effect,11 it is possible that the T106I mutation for exon 3 was 0.41 (Figure 4). The gene dosage val- impairs the enzyme activity by a similar mechanism. ues for other exons of these patients were within the The 2 patients with this mutation were suspected by normal range (0.8-1.2). These results suggest that haplotype analysis to originate from a common patient P5 had a heterozygous deletion in both exon 3 founder, although family studies would be necessary to and exon 4 and that patient P6 had a heterozygous prove this, as well as to infer the generation in which deletion in exon 3. The remaining 2 patients (P7 and the founder appeared. A notably high rate of childhood P8) exhibited gene dosage values between 0.84 and dystonia or dystonia in general is not evident in the 1.22 in all exons. geographic region where the patients live, suggesting

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Table 3. Concentrations of Neopterin and Biopterin A in Cerebrospinal Fluid of Patients P3, P4, and P5 1.2 1.0 Normal 0.8 P3 P4 P5 Value* 0.6 Mutation V206fs Possible Exons 3-4 0.4 0.2 splice deletion Gene Dosage Value site 0 123456 change Exon Age at onset, y 7 4 8 14-50 B Neopterin level, pmol/mL 8.5 3.9 8.6 22.1 ± 7.0† 1.2 Biopterin level, pmol/mL 8.2 1.3 15.7 26.4 ± 8.5† 1.0 0.8 *Normal values have been cited from Fujishiro et al.21 0.6 †Mean ± SD. 0.4 0.2 Gene Dosage Value 0 123456 that the occurrence of the mutation was not such a dis- Exon C tant event as to cause a founder effect in this region. 1.2 We also found 2 additional novel GCH1 mutations. 1.0 In 1 patient, we identified a V206fs mutation that 0.8 would terminate the translation of active-site sequences 0.6 of the GCH1 enzyme.19,20 The markedly low neopterin 0.4 0.2 and biopterin levels in the patient’s CSF proved the Gene Dosage Value 0 pathogenicity of this mutation. In the other patient, we 123456 detected a deletion of both exons 3 and 4, which is a Exon new type of genomic exon deletion, although a patient D of an English-Canadian family with a genomic exon 3 1.2 1.0 deletion has been reported to express messenger RNA 0.8 14 lacking both exons 3 and 4. The pathogenesis of this 0.6 mutation was proved by the low levels of neopterin and 0.4 0.2

biopterin in the patient’s CSF. Gene Dosage Value In 2 of the 8 patients, none of the known mutations 0 123456 was detected in any of the 6 exons or the proximal Exon splice sites of the GCH1 gene. Thus, the frequency of E detectable GCH1 mutationwas6in8.Itispossible 1.2 that these patients may have a mutation in the pro- 1.0 moter region or the untranslated region of the gene. 0.8 0.6 They did not harbor a known mutation in other genes 0.4 related to dystonia, such as tyrosine hydroxylase, 0.2 Gene Dosage Value epsilon-sarcoglycan, dopamine receptor D2, and pan- 0 123456 tothenate kinase 2. Both patients were levodopa Exon responsive and showed no particular clinical characteristics except for a relatively high age at onset (20 Figure 4. Gene dosage analysis of the 6 exons. The values between 0.8 and years and 18 years). According to a cohort study on 1.2 (boxed) were considered normal. A, Normal control. B, Patient P5. nervous and mental disorders in the Japanese popula- In patient P5, the values for exons 3 and 4 were 0.42 and 0.60, respectively. tion, DYT5 dystonia is about 1.9 times more frequent C, Patient P6. In patient P6, the value for exon 3 was 0.41. D, Patient P7. E. Patient P8. than DYT1 dystonia (74 and 40 patients among 259 patients with hereditary dystonia, respectively) (K.H. et al, unpublished data). Thus, mutation analysis of versity School of Medicine, Akita (Dr Toyoshima), De- the GCH1 gene is particularly important in Japan for partment of Pediatrics, Nagasaki Prefectural Medical conclusive diagnosis. Rehabilitation and Welfare Center for Children, Na- gasaki (Dr Matsuo), Department of Pediatrics, Kagawa Accepted for Publication: April 11, 2006. University, Takamatsu (Drs Tomoko, Yukihiko, and Author Affiliations: Division of Clinical Immunology, Yoshino), Department of Pediatrics, Tohoku University Graduate School of Medical Sciences, Kitasato Univer- School of Medicine, Sendai (Dr Yokoyama), and Depart- sity (Drs Funayama and Obata and Mr Ohta) and De- ment of Neurology, Tokushima University School of Medi- partment of Neurology, National Sagamihara Hospital (Drs cine, Tokushima (Drs Shimazu and Maeda), Japan. Funayama and Hasegawa), Kanagawa, Department of Life Correspondence: Fumiya Obata, PhD, Division of Im- Science, Graduate School of Bioscience and Biotechnol- munology, Kitasato University School of Allied Health ogy, Tokyo Institute of Technology, Tokyo (Dr Ichinose Sciences, 1-15-1 Kitasato, Sagamihara, Kanagawa 228- and Ms Urano), Department of Neurology, Akita Uni- 8555, Japan ([email protected]).

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©2006 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Author Contributions: Study concept and design: Ohta and with neopterin, biopterin, dopamine, and serotonin deficiencies and muscular Obata. Acquisition of data: Ohta, Toyoshima, Matsuo, hypotonia. Eur J Pediatr. 1984;141:208-214. 8. Furukawa Y, Kish SJ, Bebin EM, et al. Dystonia with motor delay in compound Yukihiko, Yoshino, Yokoyama, Shimazu, and Maeda. heterozygotes for GTP-cyclohydrolase I gene mutations. Ann Neurol. 1998; Analysis and interpretation of data: Ohta, Funayama, 44:10-16. Ichinose, Toyoshima, Urano, Tomoko, Hasegawa, and 9. Ichinose H, Ohye T, Matsuda Y, et al. Characterization of mouse and human GTP Obata. Drafting of the manuscript: Ohta, Tomoko, cyclohydrolase I genes: mutations in patients with GTP cyclohydrolase I deficiency. Yukihiko, Yoshino, and Obata. Critical revision of the J Biol Chem. 1995;270:10062-10071. 10. Furukawa Y, Shimadzu M, Rajput AH, et al. GTP-cyclohydrolase I gene muta- manuscript for important intellectual content: Funayama, tions in hereditary progressive and dopa-responsive dystonia. Ann Neurol. 1996; Ichinose, Toyoshima, Urano, Matsuo, Yokoyama, 39:609-617. Shimazu, Maeda, Hasegawa, and Obata. Obtained fund- 11. Hirano M, Yanagihara T, Ueno S. Dominant negative effect of GTP cyclohydro- ing: Shimazu and Maeda. Administrative, technical, and lase I mutations in dopa-responsive hereditary progressive dystonia. Ann Neurol. material support: Ohta, Funayama, Ichinose, Urano, 1998;44:365-371. Yokoyama, and Obata. Study supervision: Hasegawa and 12. Furukawa Y, Lang AE, Trugman JM, et al. Gender-related penetrance and de novo GTP-cyclohydrolase I gene mutations in dopa-responsive dystonia. Neurology. Obata. 1998;50:1015-1020. Financial Disclosure: None reported. 13. Nishiyama N, Yukishita S, Hagiwara H, Kakimoto S, Nomura Y, Segawa M. Funding/Support: This study was supported by The Re- Gene mutation in hereditary progressive dystonia with marked diurnal fluctua- search Grant (15B-2) for Nervous and Mental Disorders tion (HPD), strictly defined dopa-responsive dystonia. Brain Dev. 2000;22: from the Ministry of Health, Labor, and Welfare. S102-S106. 14. Furukawa Y, Guttman M, Sparagana SP, et al. Dopa-responsive dystonia due to a large deletion in the GTP cyclohydrolase I gene. Ann Neurol. 2000;47: REFERENCES 517-520. 15. Klein C, Hedrich K, Kabakci K, et al. Exon deletions in the GCHI gene in two of 1. Segawa M, Hosaka A, Miyagawa F, Nomura Y, Imai H. Hereditary progressive four Turkish families with dopa-responsive dystonia. Neurology. 2002;59: dystonia with marked diurnal fluctuation. Adv Neurol. 1976;14:215-233. 1783-1786. 2. Nygaard TG, Wilhelmsen KC, Risch NJ, et al. Linkage mapping of dopa- 16. Hagenah J, Saunders-Pullman R, Hedrich K, et al. High mutation rate in dopa- responsive dystonia (DRD) to chromosome 14q. Nat Genet. 1993;5:386-391. responsive dystonia: detection with comprehensive GCHI screening. Neurology. 3. Ichinose H, Ohye T, Takahashi E, et al. Hereditary progressive dystonia with marked 2005;64:908-911. diurnal fluctuation caused by mutations in the GTP cyclohydrolase I gene. Nat 17. Ohta E, Toyoshima I, Funayama M, Ichinose H, Hasegawa K, Obata F. A new mu- Genet. 1994;8:236-242. tation (Thr106Ile) of the GTP cyclohydrolase 1 gene associated with DYT5 dys- 4. Ichinose H, Ohye T, Segawa M, et al. GTP cyclohydrolase I gene in hereditary tonia (Segawa disease). Mov Disord. 2005;20:1083-1084. progressive dystonia with marked diurnal fluctuation. Neurosci Lett. 1995; 18. Fukushima T, Nixon JC. Analysis of reduced forms of biopterin in biological tis- 196:5-8. sues and fluids. Anal Biochem. 1980;102:176-188. 5. Blau N, Niederwieser A. GTP-cyclohydrolases: a review. J Clin Chem Clin Biochem. 19. Nar H, Huber R, Meining W, Schmid C, Weinkauf S, Bacher A. Atomic structure 1985;23:169-176. of GTP cyclohydrolase I. Structure. 1995;3:459-466. 6. Levine RA, Miller LP, Lovenberg W. Tetrahydrobiopterin in striatum: localiza- 20. Nar H, Huber R, Auerbach G, et al. Active site topology and reaction mechanism tion in dopamine nerve terminals and role in catecholamine synthesis. Science. of GTP cyclohydrolase I. Proc Natl Acad Sci U S A. 1995;92:12120-12125. 1981;214:919-921. 21. Fujishiro K, Hagihara M, Takahashi A, Nagatsu T. Concentrations of neopterin 7. Niederwieser A, Blau N, Wang M, Joller P, Atares M, Cardesa-Garcia J. GTP cy- and biopterin in the cerebrospinal fluid of patients with Parkinson’s disease. Bio- clohydrolase I deficiency, a new enzyme defect causing hyperphenylalaninemia chem Med Metab Biol. 1990;44:97-100.

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