©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of Mouse and Human GTP Cyclohydrolase I Genes
MUTATIONS IN PATIENTS WITH GTP CYCLOHYDROLASE I DEFICIENCY (*)

Hiroshi Ichinose (1), Tamae Ohye (1), Yoichi Matsuda (2), Tada-aki Hori (2), Nenad Blau (3), Alberto Burlina (4), Bobbye Rouse (5) (6), Reuben Matalon (5), Keisuke Fujita (1), Toshiharu Nagatsu (1)(§)

From the (1) Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-11, Japan, the (2) Division of Genetics, National Institute of Radiological Sciences, 4-9-1 Anagawa, Chiba 263, Japan, the (3) Division of Clinical Chemistry, Department of Pediatrics, University of Zürich, CH-8032 Zürich, Switzerland, the (4) Department of Pediatrics, University of Padua, 5-35128 Padua, Italy, the (5) Miami Children's Hospital, Research Institute, Miami, Florida 33155-3009, and the (6) Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas 77550

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

GTP cyclohydrolase I is the first and rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin in mammals. Previously, we reported three species of human GTP cyclohydrolase I cDNA in a human liver cDNA library (Togari, A., Ichinose, H., Matsumoto, S., Fujita, K., and Nagatsu, T. (1992) Biochem. Biophys. Res. Commun. 187, 359-365). Furthermore, very recently, we found that the GTP cyclohydrolase I gene is causative for hereditary progressive dystonia with marked diurnal fluctuation, also known as DOPA-responsive dystonia (Ichinose, H., Ohye, T., Takahashi, E., Seki, N., Hori, T., Segawa, M., Nomura, Y., Endo, K., Tanaka, H., Tsuji, S., Fujita, K., and Nagatsu, T. (1994) Nature Genetics 8, 236-242). To clarify the mechanisms that regulate transcription of the GTP cyclohydrolase I gene and to generate multiple species of mRNA, we isolated genomic DNA clones for the human and mouse GTP cyclohydrolase I genes. Structural analysis of the isolated clones revealed that the GTP cyclohydrolase I gene is encoded by a single copy gene and is composed of six exons spanning 30 kilobases. We sequenced all exon/intron boundaries of the human and mouse genes. Structural analysis also demonstrated that the heterogeneity of GTP cyclohydrolase I mRNA is caused by an alternative usage of the splicing acceptor site at the sixth exon. The transcription start site of the mouse GTP cyclohydrolase I gene and the 5`-flanking sequences of the mouse and human genes were determined. We performed regional mapping of the mouse gene by fluorescence in situ hybridization, and the mouse GTP cyclohydrolase I gene was assigned to region C2-3 of mouse chromosome 14. We identified missense mutations in patients with GTP cyclohydrolase I deficiency and expressed mutated enzymes in Escherichia coli to confirm alterations in the enzyme activity.


INTRODUCTION

GTP cyclohydrolase I (EC 3.5.4.16) catalyzes the formation of D- erythro-7,8-dihydroneopterin triphosphate from GTP. This is the first step for the de novo biosynthesis of (6 R)-(L- erythro-1`,2`-dihydroxypropyl)-2-amino-4-oxo-5,6,7,8-tetrahydropteridine (tetrahydrobiopterin) (1) . Tetrahydrobiopterin has multiple physiological functions. It acts as an essential cofactor for three aromatic amino-acid monooxygenases, i.e. phenylalanine, tyrosine, and tryptophan hydroxylases (2, 3, 4) . These enzymes are essential for synthesizing hormones and neurotransmitters such as dopamine, noradrenaline, adrenaline, and serotonin. It has also been shown that tetrahydrobiopterin acts as a cofactor for the generation of nitric oxide from arginine (5, 6) . Besides these cofactor roles, tetrahydrobiopterin has been suggested to be involved in the proliferation and growth of erythroid cells (7) .

The de novo biosynthesis of tetrahydrobiopterin is thought to be regulated by the activity of GTP cyclohydrolase I (1) , and the GTP cyclohydrolase I activity increases in response to various stimuli. For example, the addition of interferon- (8) , phytohemagglutinin (9) , or kit ligand (10) greatly induces the GTP cyclohydrolase I activity. It is thus of great importance to clarify the regulatory mechanism(s) underlying the inducible expression of the GTP cyclohydrolase I gene.

There have been several reports on tetrahydrobiopterin-dependent hyperphenylalaninemia caused by a deficiency in GTP cyclohydrolase I (11, 12, 13) . Hyperphenylalaninemia caused by GTP cyclohydrolase I deficiency shows more severe symptoms than that caused by a defect in phenylalanine hydroxylase because a deficiency in GTP cyclohydrolase I impairs the biosynthesis of both catecholamines and serotonin due to the lack of tetrahydrobiopterin. Patients show severe retardation of development, severe muscular hypotonia of the trunk and hypertonia of the extremities, convulsions, and frequent episodes of hyperthermia without infections (11, 12, 13) .

Very recently, we found that the gene for GTP cyclohydrolase I is causative for hereditary progressive dystonia with marked diurnal fluctuation (HPD),() also known as DOPA-responsive dystonia (DRD) (14) . This disease is inherited as an autosomal dominant trait with a low penetrance. We found that HPD/DRD patients have genetic defects in the GTP cyclohydrolase I gene only in one allele and that their GTP cyclohydrolase I activity in mononuclear blood cells is reduced to <20% of that of normal individuals. If only one allele carries the mutant gene, the enzyme activity would be expected to be decreased to about half of the control value. We assumed that the expression of GTP cyclohydrolase I in HPD/DRD patients would be lower than in normal individuals. To elucidate the etiology of HPD/DRD in more detail, we undertook molecular characterization of the GTP cyclohydrolase I gene.

GTP cyclohydrolase I cDNA was first isolated from a rat liver cDNA library (15) . Previously, we reported the isolation of three GTP cyclohydrolase I cDNA clones from human liver (16) . All three cDNAs were identical at their central and 5`-regions, but diverged at their 3`-ends. We designated these three cDNAs as types 1-3. Type 1 cDNA, which has the longest coding region, consisting of 250 amino acids, corresponds to rat (15) and mouse (17) cDNAs.

For elucidation of the mechanisms regulating the expression of the GTP cyclohydrolase I gene and the mechanism generating the molecular heterogeneity of its mRNA, structural information on the gene is indispensable. Furthermore, recent advances in murine developmental biology have made it possible to knock out a specific gene in mice. Mice having a defect in the GTP cyclohydrolase I gene would be useful for analyzing physiological roles for tetrahydrobiopterin and as an animal model of hyperphenylalaninemia caused by GTP cyclohydrolase I deficiency or HPD/DRD.

In this study, we isolated and characterized the mouse and human GTP cyclohydrolase I genes. Furthermore, we assigned the chromosomal location of mouse GTP cyclohydrolase I. This is the first report on the genomic structure of the GTP cyclohydrolase I gene in mammals.


MATERIALS AND METHODS

Screening of a Genomic Library and Analysis of Phage Clones

A mouse genomic library, constructed in Lambda FixII phage vectors (Stratagene), was screened by use of a mouse GTP cyclohydrolase I cDNA clone (17) labeled with [-P]dCTP. Human genomic DNA libraries, constructed in Charon 4A (donated by Dr. Tom Maniatis, Harvard University) and in EMBL3 (LI 018; donated by the Japanese Cancer Research Resources Bank), were screened with a human GTP cyclohydrolase I cDNA clone (16) used as a probe. DNAs were isolated from the purified phage plaques following standard protocols (18) , and the fragments of interest were subcloned into pBluescript KS (Stratagene) or pUC119 vectors for further structural analysis. The nucleotide sequences of the clones were determined by the dideoxy chain termination method (19) using Sequenase (United States Biochemical Corp.).

DNA Blot Hybridization Analysis

Mouse genomic DNA (10 µg) was digested with EcoRI, HindIII, and SacI; separated according to size on a 0.8% agarose gel; and capillary-blotted onto a nylon membrane (Hybond-N, Amersham Corp.). This membrane was hybridized with a P-labeled full-length cDNA clone of mouse GTP cyclohydrolase I (17) in a solution consisting of 6 SSC (1 SSC = 0.15 M NaCl and 0.015 M sodium citrate), 5 Denhardt's solution, 0.5% SDS, and 0.1 mg/ml salmon sperm DNA. After hybridization at 65 °C overnight, the membrane was washed twice with 2 SSC containing 0.05% SDS at room temperature for 5 min and twice with the same solution at 42 °C for 15 min.

Transcription Initiation Site Mapping

Total RNA was extracted from mouse brain with guanidinium thiocyanate followed by centrifugation in cesium chloride solutions. The transcription initiation site was determined by both primer extension analysis and reverse transcription-polymerase chain reaction (RT-PCR) analysis.

In the primer extension experiment, mouse brain poly(A)RNA (1.5 µg) was annealed to P-end-labeled primer m-42R (5`-AACAAGCGCTGCGGCTCAGCT-3`) in 0.25 M KCl by heating at 65 °C for 1 h, followed by incubation at room temperature for 1.5 h. The annealed primer was extended at 45 °C for 1 h in a reaction mixture containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl, 10 mM dithiothreitol, 0.25 mM dNTPs, 110 units of RNase inhibitor, and 400 units of reverse transcriptase (SuperScript II, Life Technologies, Inc.). Then the sample was precipitated by ethanol and analyzed on a 7 M urea, 6% acrylamide sequencing gel. The DNA fragment containing exon 1 was sequenced with primer m-42R at the same time.

For RT-PCR experiments, total RNA from mouse brain was reverse-transcribed with random hexamer and amplified using primer m-42R as an antisense primer and primer m-189F, m-131F, or m-122F as a sense primer. Their sequences and positions on genomic DNA are shown in Fig. 5 . To remove a trace amount of genomic DNA contaminating the RNA preparation, the total RNA was subjected to digestion with RNase-free DNase I (Boehringer Mannheim) prior to the reverse transcription reaction. Amplification for 30 cycles was carried out as follows: denaturation at 94 °C for 30 s and annealing and extension at 60 °C for 1 min. The amplified DNA fragments were electrophoresed on 4% NuSieve GTG agarose (FMC Corp. BioProducts).


Figure 5: Sequence of the 5`-flanking region of the mouse GTP cyclohydrolase I gene. The nucleotide sequence is numbered with the first base of the ATG initiation codon designated as position +1. The location of the transcription initiation site determined by RT-PCR and primer extension analyses is indicated by the asterisk. Primers used for RT-PCR and primer extension are designated by half-arrows. An AT-rich region and a CCAAT sequence are boxed. An H1 box and consensus sequences for IBP-1b- and GT-2B-binding proteins are underlined.



Chromosomal Localization of the GTP Cyclohydrolase I Gene by Fluorescence in Situ Hybridization

The direct R-banding fluorescence in situ hybridization method (20) was used for chromosomal localization of the mouse GTP cyclohydrolase I gene. Mouse lymphocyte cultures and preparation of R-banded chromosomes were performed as described previously (20) .

The chromosomes on slides were hardened at 65 °C for 2 h, denatured at 70 °C in 70% formamide in 2 SSC, and then dehydrated in 70/85/100% ethanol series at 4 °C. The HindIII-digested mouse genomic DNA fragment (3.5 kb in length) containing exon 2 was labeled by nick translation with biotinylated 16-dUTP (Boehringer Mannheim) following the manufacturer's protocol. The labeled DNA fragments were ethanol-precipitated and then denatured in 100% formamide at 75 °C for 10 min. The denatured probe was mixed with an equal volume of hybridization solution to make final concentrations of 50% formamide, 2 SSC, 10% dextran sulfate, and 1 mg/ml bovine serum albumin (Sigma). Twenty microliters of the mixture containing 250 ng of probe DNA was put on the denatured chromosomes, and the slides were then covered with Parafilm and incubated overnight at 37 °C, after which they were washed for 20 min in 50% formamide in 2 SSC at 37 °C and in 2 SSC and 1 SSC for 20 min each at room temperature. After the slides had been rinsed in 4 SSC, the chromosomes were heat-incubated under a coverslip with fluoresceinated avidin (Vector Labs, Inc.) at a 1:500 dilution in 1% bovine serum albumin, 4 SSC for 45 min at 37 °C. The slides were then washed with 4 SSC, 0.1% Nonidet P-40 in 4 SSC and with 4 SSC for 10 min each on a shaker. After the excess liquid had been drained from the slides, the chromosomes were finally stained with 0.75 µg/ml propidium iodide.

Excitation was carried out at a wavelength of 450-490 nm (Nikon filter set B-2A), and a near 365-nm filter (UV-2A) was used for observation. Kodak Ektachrome ASA 100 films were used for microphotography.

Case Reports

Patient N. R. is the second child of healthy nonconsanguineous parents. Pregnancy and delivery at term were uneventful; breast feeding was tolerated well, and the Guthrie phenylketonuria screening test performed at the age of 4 days was normal. The child was diagnosed as hyperphenylalaninemic at the age of 5 months, presenting progressive neurological deterioration, including severe hypotonia and uncoordinated and generalized movements (Parkinson-like symptoms). Metabolic studies including analysis of pterins in urine and cerebrospinal fluid as well as measurement of GTP cyclohydrolase I activity in a liver biopsy led to the diagnosis of GTP cyclohydrolase I deficiency () (21) . Patient N. R. is still living and is in good health (21) .

Patient M. K., a female born near term weighing 2.7 kg, was identified by newborn screening as having phenylketonuria with blood phenylalanine values >2400 µmol/liter (22) . In the first week of life, the baby developed feeding problems, poor sucking, and poor muscle tone. At the age of 6 months, in spite of good control of blood phenylalanine levels, the baby was found to be delayed in development. By the age of 2 years, the child was unable to walk and developed seizures and choreoathetosis. Urinary pterins showed a profound deficiency in neopterin and biopterin (). The same deficiency was found in the plasma (data not shown). Administration of tetrahydrobiopterin, 5-hydroxytryptophan, and L-DOPA/carbidopa reduced the blood phenylalanine level and improved the choreoathetosis. Patient M. K. died at the age of 10 years. Diagnosis was confirmed post-mortem by the measurement of the enzyme activity in the liver.

Mutation Analysis

Genomic DNA was extracted from primary skin fibroblasts. We amplified exons for GTP cyclohydrolase I including splicing junctions using PCR on genomic DNA. Primer sequences used for amplification of exons were as follows: exon 1, 5`-GTTTAGCCGCAGACCTCGAAGCG-3` and 5`-GAGGCAACTCCGGAAACTTCCTG-3`; exon 2, 5`-GTAACGCTCGCTTATGTTGACTGTC-3` and 5`-ACCTGAGATATCAGCAATTGGCAGC-3`; exon 3, 5`-AGATGTTTTCAAGGTAATACATTGTCG-3` and 5`-TAGATTCTCAGCAGATGAGGGCAG-3`; exon 4, 5`-GTCCTTTTTGTTTTATGAGGAAGGC-3` and 5`-GGTGATGCACTCTTATAATCTCAGC-3`; exon 5, 5`-GTGTCAGACTCTCAAACTGAGCTC-3` and 5`-TCACTTCTAGTGCACCATTATGACG-3`; and exon 6, 5`-ACCAAACCAGCAGCTGTCTACTCC-3` and 5`-AATGCTACTGGCAGTACGATCGG-3`. PCR amplification was performed in a reaction volume of 25 µl containing 100 ng of genomic DNA, 10 pmol of each primer, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl, 5% dimethyl sulfoxide, 0.2 unit of Perfectmatch (Stratagene), and 1.25 units of Taq polymerase (Perkin-Elmer). After having been heated at 94 °C for 3 min, the reaction mixture was cycled according to the following program: 30 cycles for 30 s at 94 °C and for 1 min at 60 °C and a final extension at 60 °C for 6 min. The PCR products were sequenced directly with the same primers used for amplification by use of an automatic DNA sequencer (Perkin-Elmer Model 373A) and a DyeDeoxy Terminator Cycle sequencing kit.

Site-directed Mutagenesis and Expression of GTP Cyclohydrolase I in Escherichia coli

To perform oligonucleotide-directed in vitro mutagenesis, we followed the manufacturer's protocol for the Sculptor in vitro mutagenesis system (Amersham Corp.). We used plasmid Bluescript KS containing human GTP cyclohydrolase I cDNA (hGCH1) (16) as a template for construction of mutant GTP cyclohydrolase I cDNAs by site-directed mutagenesis. Although the hGCH1 cDNA clone lacked the first 25 bases corresponding to nine amino acids from the starting methionine, the first nine amino acids in the human sequence do not seem to be important for the activity since mouse and rat enzymes lack these nine residues. The mutagenesis primers were 5`-GCAACACACATATGTATGGTAATG-3` for the generation of the M211I mutation and 5`-GTTCAGGAGCACCTTACAAAAC-3` for the generation of R184H (the mutated nucleotides are underlined). The integrity of the newly constructed mutant cDNAs was checked by sequencing the plasmids.

Wild-type and mutant cDNAs were introduced into the EcoRI site of a pGEX-2T vector (Pharmacia Biotech Inc.), which can express a foreign gene as the fusion protein with glutathione S-transferase. E. coli strain BL21 cells carrying each plasmid were cultured at 37 °C to late-log phase, and the expression of the desired protein was induced by the addition of isopropyl--D-thiogalactopyranoside. Expressed proteins were analyzed by SDS-10% polyacrylamide gel electrophoresis and stained with Coomassie Brilliant Blue R-250. GTP cyclohydrolase I activities were measured as described previously (14) with crude homogenate used as an enzyme source.

Miscellaneous

Proteins were determined by the method of Bradford (23) with bovine -globulin used as a standard. Student's t test was used for statistical evaluation of the mutated enzyme activity expressed in E. coli.


RESULTS

Isolation and Characterization of Mouse GTP Cyclohydrolase I Genomic Clones

Using the cloned mouse GTP cyclohydrolase I cDNA (17) as a probe, we isolated five overlapping genomic DNA clones by screening a mouse genomic library constructed in FixII (mgGCH1-5) (Fig. 1). Structural analysis of these clones revealed that the complete mouse GTP cyclohydrolase I gene is 32 kb in length and consists of a total of six exons. We sequenced the exons and their splice sites. As shown in Fig. 2, each exon/intron boundary conformed perfectly to the GT/AG rule (24) .


Figure 1: Structure of the mouse GTP cyclohydrolase I gene. The structure of the mouse GTP cyclohydrolase I gene is depicted at the top. The six exons are indicated by closed boxes and introns by open boxes. Five isolated clones (mgGCH1-5) covering >30 kb of the mouse GTP cyclohydrolase I gene are depicted below the structure of the gene. EcoRI restriction sites ( E) are indicated.




Figure 2: Exon/intron boundary sequences of the mouse GTP cyclohydrolase I gene. Exon sequences are written in upper-case letters and intron sequences in lower-case letters. Sequences are numbered with the first base of the ATG initiation codon designated as position +1 (nucleotides within introns are not numbered). Sizes of both exons and introns are indicated. AATAAA as a polyadenylation signal (24) is underlined. b, bases.



Genomic DNA Blot Hybridization Analysis

To determine the copy number of the GTP cyclohydrolase I gene, we performed genomic DNA blot hybridization analysis. High molecular weight DNA samples were digested with the restriction enzymes EcoRI, HindIII, and SacI; separated electrophoretically on an agarose gel; and then transferred to a nylon membrane. The membrane was then hybridized with the mouse cDNA clone (Fig. 3). The sizes of bands showed good coincidence with those predicted from the restriction enzyme site mapping of the genomic DNA clones. This result suggests that GTP cyclohydrolase I is specified by a single copy gene.


Figure 3: Southern hybridization analysis of mouse genomic DNA. Ten micrograms of mouse genomic DNA was digested with EcoRI, HindIII, or SacI. After electrophoresis on a 0.8% agarose gel, each digest was transferred to a nylon membrane and hybridized with P-labeled mouse GTP cyclohydrolase I cDNA as a probe.



Determination of the Transcription Start Site

The transcription start site was determined by primer extension analysis and was confirmed by RT-PCR analysis as described under ``Materials and Methods.'' Primer extension analysis was performed with a P-end-labeled oligonucleotide (m-42R) located in exon 1 and with poly(A)RNA from mouse liver. As shown in Fig. 4 A, this experiment yielded a major primer-extended product of 125 nucleotides with minor products ranging from 123 to 126 nucleotides. The length of the fragment mapped the initiation site around the guanine nucleotide at position 164.


Figure 4: Determination of the transcription start site of the mouse GTP cyclohydrolase I gene. The transcription initiation site was determined by primer extension ( A) and RT-PCR ( B) analyses as described under ``Materials and Methods.'' A, primer extension analysis was performed with antisense primer m-42R and poly(A)RNA from mouse liver. The reverse transcription reaction was performed with (+) or without () reverse transcriptase. Asterisks indicate the positions of extension products. The locations of the extension stop points were determined by parallel lanes containing a dideoxynucleotide-terminated sequence. B, total RNA from mouse liver was reverse-transcribed by use of random hexamer oligonucleotides. The resulting cDNA ( lanes 1-3) was subjected to PCR amplification using primer m-42R as an antisense primer and primer m-122F ( lanes 1 and 4), m-131F ( lanes 2 and 5), or m-189F ( lanes 3 and 6) as a sense primer. A cloned genomic DNA containing the 5`-flanking region was also subjected to PCR amplification using the same primer sets ( lanes 4-6) to monitor PCR. Lane M contains an HaeIII-digested X174 marker.



To confirm this result, we performed RT-PCR analysis using m-42R as an antisense primer and m-122F, m-131F, or m-189F as a sense primer (Fig. 4 B). Total RNA from mouse liver generated amplified products of the expected size with primers m-122F and m-131F, whereas primer m-189F showed no band (Fig. 4 B, lanes 1-3). These three primers correctly amplified the plasmid DNA containing exon 1 (Fig. 4 B, lanes 4-6). These results indicate that the 5`-end of the mRNA is present between primers m-131F and m-189F. This agreed with the result of the primer extension analysis. On the basis of these two results, the transcription start site of mouse GTP cyclohydrolase I mRNA was determined to be around guanine at position 164, ranging from cytidine at position 162 to adenine at position 165.

DNA Sequence Analysis of the 5`-Flanking Region of the Mouse GTP Cyclohydrolase I Gene

The nucleotide sequence of the 5`-flanking region was determined to 620 base pairs upstream from the transcription start site and is shown in Fig. 5. This sequence has been submitted to the DDBJ data base. No obvious TATA box was observed in the promoter region. Instead, an AT-rich putative promoter motif (ATAAAAA) or a CCAAT box was present just upstream or 50 base pairs upstream from the transcription start site, respectively. In addition, H1 box (25) -, IBP-1b (26) -, and GT-2B (27) -binding consensus sequences were found.

Chromosomal Mapping of the Mouse GTP Cyclohydrolase I Gene

The direct R-banding fluorescence in situ hybridization method (20) was performed for chromosomal localization of the mouse GTP cyclohydrolase I gene, with a HindIII-digested genomic DNA fragment (3.5 kb in length) containing exon 2 used as a probe. The signals were localized to the region of C2-3 on chromosome 14 (data not shown). The standard G- and R-banded karyotypes have been reported by Matsuda et al. (20) .

Isolation and Characterization of Human GTP Cyclohydrolase I Genomic Clones

We previously isolated three species of cDNA encoding GTP cyclohydrolase I from a human liver cDNA library (16) . All three cDNAs were identical at their central and 5`-regions, but diverged at their 3`-ends. To clarify the mechanism(s) that generate this heterogeneity of GTP cyclohydrolase I mRNA, we analyzed the human GTP cyclohydrolase I gene structure. Using a cDNA clone as a probe, we isolated three genomic DNA clones from genomic DNA libraries constructed in Charon 4A (hgGCH6) or EMBL3 (hgGCH5 and hgGCH10) (Fig. 6). Clone hgGCH5 contained exon 1; hgGCH10 contained exon 2; and hgGCH6 contained exons 4-6. Exon 3 was missing in these clones, although the clones encompassed >30 kb of human chromosomal DNA. To search for exon 3, we used PCR to amplify genomic DNA from the end of hgGCH10 to exon 3. PCR amplified a DNA fragment with a length of 1 kb. Sequence analysis of the fragment revealed that the correct DNA region was amplified. The length between exons 3 and 4 was determined by PCR. The recently developed long PCR technology enabled us to amplify the entire DNA region between exons 3 and 4, which showed a length of 9 kb.


Figure 6: Structure of the human GTP cyclohydrolase I gene. Isolated clones (hgGCH1-3) of the human GTP cyclohydrolase I gene are depicted. Coding regions are indicated by closed boxes and untranslated regions by open boxes. Introns are represented by thin horizontal lines. EcoRI restriction sites ( E) are indicated. The region amplified by PCR is shown by a dashed line.



Structural analysis of these clones revealed that the human GTP cyclohydrolase I gene consists of six exons. We sequenced exon/intron boundaries, and these sequences have been submitted to GenBank. Break points between exons were completely identical to those of the mouse gene (Fig. 7). Each exon/intron boundary conformed to the GT/AG rule (24) .


Figure 7: Exon/intron boundary sequences of the human GTP cyclohydrolase I gene. Exon sequences are written in upper-case letters and intron sequences in lower-case letters. Sequences are numbered with the first base of the ATG initiation codon designated as position +1 (nucleotides within introns are not numbered). Sizes of both exons and introns are indicated. ATTAAA as a polyadenylation signal (24) is underlined. b, bases.



A marked difference between the human and mouse GTP cyclohydrolase I genes was observed in the last exon (exon 6). We also isolated human cDNAs from a human pheochromocytoma cDNA library and sequenced a cDNA of 2.4 kb in length. The cDNA clone had an open reading frame corresponding to type 1 (a common type in human, rat, and mouse) and an extremely long 3`-noncoding region (2 kb) with a poly(A)stretch. The entire 3`-noncoding region was encoded in exon 6. The nucleotide sequence of human exon 6 is shown in Fig. 8 and has been submitted to the DDBJ data base.


Figure 8: Entire sequence of exon 6. Nucleotides are numbered above the DNA sequence with the first base of the ATG initiation codon designated as position +1. ATTAAA as a polyadenylation signal (24) is underlined.



Analysis of the genomic DNA structure revealed that three cDNAs diverged just after the break point between exons 5 and 6. We found the type 2 cDNA sequence at the middle of exon 6. The sequence at the 3`-splice site of intron 5 was TATTTTGTAGAC, while the sequence around the splice site for type 2 mRNA was TCATTTTCAGGT. Thus, a splicing between the 5`-splice site of intron 5 (ACGTAAGTCTGC) and the 3`-splice site of intron 5 produces type 1 mRNA, whereas a splicing between the 5`-splice site of intron 5 and the middle of exon 6 generates type 2 mRNA. On the other hand, the sequence of intron 5 completely matched the divergent sequence of type 3 cDNA, indicating that intron 5 is not spliced out in the transcript corresponding to type 3 cDNA. The alternative splicing mechanism suspected to produce heterogeneity of GTP cyclohydrolase I mRNAs is illustrated in Fig. 9.


Figure 9: Mechanism generating human GTP cyclohydrolase I mRNA heterogeneity. See ``Results'' for details.



DNA Sequence Analysis of the 5`-Flanking Region of the Human GTP Cyclohydrolase I Gene

The nucleotide sequence of the 5`-flanking region of exon 1 was determined to 600 base pairs upstream from the first exon and was compared with that of the mouse GTP cyclohydrolase I gene (Fig. 10). Both human and mouse 5`-flanking sequences have been submitted to DDBJ. Several highly conserved regions are boxed in Fig. 10. The AT-rich putative promoter motif and the CCAAT box found in the mouse gene were conserved in the human GTP cyclohydrolase I gene. A consensus sequence for the binding of GT-2B (27) was found in the human gene as well as in the mouse gene. The IBP-1b and H1 box consensus sequences found in the mouse gene were not present in the human gene, whereas a leader-binding protein-1 (28) -binding sequence motif ((A/T)CTGG, positions 479 to 475), a T-antigen-binding motif (positions 277 to 272), a TGGCA box (positions 675 to 671), and an SP-1 consensus sequence (positions 245 to 236) were present in the human gene.


Figure 10: Comparison of the 5`-flanking regions of the human and mouse GTP cyclohydrolase I genes. Identical nucleotides are shaded. Gaps (indicated by dashes) were introduced to obtain maximum homology. Highly conserved regions are boxed. The transcription start site in mouse GTP cyclohydrolase I is indicated by the asterisk. AT-rich and CAAT motifs and a binding consensus sequence for GT-2B (27) are underlined.



Mutations in the GTP Cyclohydrolase I Gene in Patients with GTP Cyclohydrolase I Deficiency

To search for mutations in the coding region of the GTP cyclohydrolase I gene, we amplified exons including splicing junctions from genomic DNA from the patients by PCR. Amplified DNA fragments were directly sequenced with an automated DNA sequencer.

We examined two patients with GTP cyclohydrolase I deficiency (patients N. R. and M. K.) and normal individuals. The patients had missense mutations in both alleles (data not shown). Patient N. R. had an A nucleotide instead of the G nucleotide found in normal subjects, and this mutation resulted in an amino acid substitution of methionine with isoleucine at position 211 (M211I). Patient M. K. showed a transition from G to A, and the arginine residue was substituted with a histidine residue at position 184 (R184H). Both patients were homozygous in terms of the mutations, and no other mutations were found in the coding region of their GTP cyclohydrolase I gene. These substitutions affect highly conserved amino acid residues of GTP cyclohydrolase I.

Expression of Mutated Enzymes in E. coli

To prove that the amino acid changes found in the patients result in functional alterations, we expressed the wild-type and mutant proteins and analyzed their GTP cyclohydrolase I activity. Expression in E. coli was carried out with the glutathione S-transferase gene fusion system. This system expresses the introduced gene as a fusion protein with glutathione S-transferase (molecular mass of glutathione S-transferase is 26,000 Da). Since human GTP cyclohydrolase I, composed of 250 amino acids, has a calculated molecular mass of 27,903 Da, the fusion protein is expected to be 54,000 Da. Expression of foreign genes was examined by SDS-polyacrylamide gel electrophoresis (Fig. 11 A). Obvious extra bands at a molecular mass of 54,000 Da appeared in E. coli extracts harboring the plasmid containing wild-type or mutant GTP cyclohydrolase I genes (M211I and R184H). The amount of expressed proteins was similar as judged by Coomassie Brilliant Blue R-250 staining. Then we measured the GTP cyclohydrolase I activities in the crude homogenates. E. coli extracts harboring wild-type cDNA showed a high GTP cyclohydrolase I activity, whereas E. coli extracts harboring the expression vector without a cDNA insert (pGEX-2T) had relatively low endogenous activity. Both the M211I and R184H substitutions completely abolished the increase in the GTP cyclohydrolase I activity shown with wild-type cDNA (Fig. 11 B). These results demonstrate that the M211I and R184H mutations are not simply polymorphisms.


Figure 11: Overexpression of wild-type or mutant GTP cyclohydrolase I in E. coli. A, SDS-polyacrylamide gel electrophoresis. E. coli strain BL21 harboring the pGEX-2T expression vector alone ( lane 1) or with wild-type ( lane 2), M211I mutant ( lane 3), or R184H mutant ( lane 4) GTP cyclohydrolase I cDNA was induced with isopropyl--D-thiogalactopyranoside. A crude protein fraction was prepared, and 20 µg of total protein was separated on each lane. The gel was stained with Coomassie Brilliant Blue R-250. The arrowhead indicates the 54-kDa GTP cyclohydrolase I protein fused with glutathione S-transferase (26 kDa). B, GTP cyclohydrolase I activities in crude E. coli BL21 extracts harboring the expression vector alone ( pGEX-2T) or with wild-type GTP cyclohydrolase I cDNA ( wild), M211I mutant cDNA, or R184H mutant cDNA. Values are represented as means ± S.E. ***, p < 0.001.




DISCUSSION

We determined the entire structures of the mouse and human GTP cyclohydrolase I genes and the chromosomal location of mouse GTP cyclohydrolase I. This is the first demonstration of the genomic structure of any vertebrate GTP cyclohydrolase I enzyme.

Togari et al. (16) reported the isolation of three types of cDNAs for human GTP cyclohydrolase I, designated as types 1-3, from a human liver cDNA library. Recently, Gütlich et al. (29) confirmed the presence of both type 1 and 2 cDNAs in their human liver cDNA library. They showed that only type 1 cDNA encodes an active enzyme and that proteins corresponding to type 2 and 3 cDNAs have no GTP cyclohydrolase I activity. The present analysis of genomic DNA revealed that these three types of cDNAs are produced from a single gene. An alternative usage of the splicing acceptor site in exon 6 generates type 2 cDNA, whereas type 3 cDNA contains the fifth intron. Although we described the presence of all three mRNAs in human liver in a previous paper (16) , there is a possibility that type 3 cDNA may be derived from an immature mRNA that has introns to be spliced out.

On the other hand, Gütlich et al. (30) observed, by RNA blot hybridization analysis, that two species (1.4 and 3.6 kb) of GTP cyclohydrolase I mRNA exist in rat and mouse tissues, but they detected only one species (3.6 kb) in human-derived cells. From our analysis of genomic DNA, a full-length human type 1 mRNA should be 3 kb in length. Probably the longer species of mRNA (3.6 kb) observed by Gütlich et al. (30) in their blot hybridization analysis corresponds to type 1 mRNA because estimation of size by RNA blot hybridization using 18 S and 28 S ribosomal RNAs is not accurate. Since type 2 mRNA is shorter than type 1 mRNA by up to 1089 bases, type 2 mRNA should be distinguishable from type 1 mRNA by RNA blot hybridization. Thus, the result of RNA blot hybridization analysis suggests that the amount of type 2 mRNA is very low compared with that of type 1 mRNA.

When we compared the gene structure of the mouse and human GTP cyclohydrolase I enzymes, we considered it noteworthy that the length of the sixth exon is quite different. The human sixth exon is 2127 base pairs in length, but the corresponding mouse exon has only 296 base pairs. Although no sequence information on longer species of rodent cDNA has yet been reported, alternative polyadenylation may produce longer species of rodent mRNA as observed in RNA blot hybridization analysis by Gütlich et al. (30) .

It is well known that GTP cyclohydrolase I is induced by various stimuli, such as interferon- (8, 31) , bacterial lipopolysaccharide (32) , and kit ligand (10) . Here we found a sequence similar to the IBP-1-binding site in the upstream region of the mouse GTP cyclohydrolase I gene. IBP-1 is a DNA-binding factor induced by treatment of HeLa cells with interferon- and is thought to be involved in the response to interferon- (26) . Recently, extensive studies were performed on the mechanism of transcriptional regulation by interferon, and these defined the DNA sequence required for activation of transcription by interferon-, the -activated site (33) . So far, we have found no typical -activated site-like sequence in the 5`-flanking region of the mouse or human GTP cyclohydrolase I gene. The molecular mechanism for transcriptional regulation of GTP cyclohydrolase I remains to be clarified.

Genetic defects in patients with GTP cyclohydrolase I deficiency were characterized for the first time in more detail. Analysis of the genomic structure of GTP cyclohydrolase I enabled us to determine mutations in patients with GTP cyclohydrolase I deficiency or HPD/DRD. Here we demonstrated that patients with GTP cyclohydrolase I deficiency are homozygous for a defect in the GTP cyclohydrolase I gene, whereas HPD/DRD patients are known to be heterozygous. In HPD/DRD patients, we had found three missense mutations (R88W, D134V, and G201E) and a 2-base insertion that shifts the reading frame just after the initiating methionine (14) . We had expressed the mutated enzymes with the R88W or G201E mutation in the same expression system as employed in the present study. Transfection with cDNAs prepared from patients with GTP cyclohydrolase I deficiency as well as from HPD/DRD patients failed to give the increase in the GTP cyclohydrolase I activity seen in the bacteria transfected with wild-type cDNA, indicating that the mutated enzymes have practically no catalytic activity. These results indicate that there is no qualitative difference between the mutations in GTP cyclohydrolase I deficiency and HPD/DRD. Since GTP cyclohydrolase I deficiency is a recessive disease and both alleles of the gene are mutated as demonstrated in this paper, patients with GTP cyclohydrolase I deficiency have no detectable amount of the enzyme activity. On the other hand, autosomal dominant inheritance with low penetrance is shown in HPD/DRD. Patients with HPD/DRD carry a mutant gene only in one allele, and they have little but some GTP cyclohydrolase I activity. Therefore, the difference in the level of the enzyme activity would explain the differing clinical presentations of these disorders, although mutations of both disorders reduced the activity of mutant proteins to zero. GTP cyclohydrolase I forms dihydroneopterin triphosphate from GTP through a very complex mechanism (1) , and the amino acid sequence of the enzyme is highly conserved among different species. Thus, various amino acid substitutions would be expected to result in a loss of the enzyme activity.

Previously, we reported the chromosomal locus for human GTP cyclohydrolase I to be at 14q22.1-q22.2 (14) . In the present study, we assigned mouse GTP cyclohydrolase I to the C2-3 region of chromosome 14. This region of the mouse chromosome corresponds to human chromosome 14. This result confirmed the localization of the human gene.

There is a mutant mouse (hph-1) that is thought to have a defect in GTP cyclohydrolase I (34, 35) . This mouse was generated by treatment with ethylnitrosourea, and the selected hph-1 mutant was screened for hyperphenylalaninemia by the Guthrie assay. But the GTP cyclohydrolase I activity in hph-1 can be detected later in life, i.e. adult mice (34) , and the phenotype of hph-1 mice is far from that of GTP cyclohydrolase I deficiency seen in man. Human GTP cyclohydrolase I deficiency results in severe symptoms, such as severe retardation of development, severe muscular hypotonia of the trunk and hypertonia of the extremities, and convulsions (11, 12, 13) . On the other hand, hph-1 mice show normal development and no sign of illness except for hyperphenylalaninemia. The mutated locus for hph-1 had been assigned to the C2-3 region of chromosome 14 (36) . This suggests that the genetic defect in the hph-1 mutant mouse would be in the structural gene or cis-acting element for GTP cyclohydrolase I itself, but not in its regulatory gene. The reason for the species difference in the phenotypes of hph-1 mice and GTP cyclohydrolase I deficiency in man is not clear. The metabolism of tetrahydrobiopterin may be different between man and mouse. It is well known that in mammals, except primates, no neopterin can be detected in significant amounts. This would be due to the rate-limiting activity of GTP cyclohydrolase I. In man, the rate-limiting step may be the conversion of dihydroneopterin triphosphate to 6-pyruvoyltetrahydropterin by the enzyme 6-pyruvoyltetrahydropterin synthase. Molecular characterization of the defect in the hph-1 mouse will help to explain the species difference in the phenotype caused by a defect in GTP cyclohydrolase I.

  
Table: Biochemical data on two GTP cyclohydrolase I-deficient patients



FOOTNOTES

*
This work was supported by a grant-in-aid for specially promoted research from the Ministry of Education, Science, and Culture of Japan; by grants-in-aid from the Ministry of Health and Welfare of Japan, the Japan Intractable Diseases Research Foundation, and the Fujita Health University; and Grant 31-33897.32 from the Swiss National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequences reported in this paper have been submitted to the GSDB, DDBJ, GenBank/EMBL, and NCBI nucleotide sequence data bases with accession numbers D38601-D38603 (sequences of the mouse and human 5`-flanking regions and the sequence of the human sixth exon) and U19256-U15259 (sequences of human exons including introns).

§
To whom correspondence should be addressed. Tel.: 81-562-93-9391; Fax: 81-562-93-8831.

The abbreviations used are: HPD, hereditary progressive dystonia with marked diurnal fluctuation; DRD, DOPA-responsive dystonia; RT-PCR, reverse transcription-polymerase chain reaction; kb, kilobase(s).


ACKNOWLEDGEMENTS

We are grateful to Takahide Nomura, Nakao Iwata, Machiyo Shirakura, and Minae Takeno for help in this research.


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