Human Tyrosine Hydroxylase Gene Research Articles

Exon 3 of tyrosine hydroxylase gene: lack of association with Japanese schizophrenic patients.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine (DA) biosynthesis. Exon 3 of the human TH gene encodes the sequence from Ser31 to Glu104 of type 1 enzyme, which contains the critical parts for regulation of the catalytic activity. The amino acid residues Gly36-Arg37-Arg38 were identified as a key sequence for DA to exert its inhibitory effect on catalytic activity. Therefore, we screened the nucleotide sequences of exon 3 from 201 Japanese patients with schizophrenia to explain the elevation in the synaptic or presynaptic DA concentrations in the schizophrenic brain, based on the hypothesis that any mutation changing the amino acid sequence Gly36-Arg37-Arg38 would result in the elevation of DA synthesis, due to a reduced inhibitory effect of DA on the catalytic activity. However, no mutated sequences of exon 3 and both exon-intron boundaries were detected in any of the patients examined. Polymorphisms generating Val81 and Met81 were compared of the distributions of genotype and allele between the patients and 175 Japanese healthy controls, which did not suggest an association between the polymorphism and schizophrenia. These results indicate that exon 3 of the human TH gene lacks association with schizophrenia in Japanese patients.

An association of the polymorphic repeat of tetranucleotide (TCAT) in the first intron of the human tyrosine hydroxylase gene with schizophrenia in a Japanese sample.

Tyrosine hydroxylase (TH) is the rate limiting enzyme in the synthesis of dopamine and norepinephrine. A polymorphic repeat of the tetranucleotide (TCAT) in the first intron of the TH gene may behave as a regulatory element for the gene expression. Allelic fragments of the tandem repeat were typed by a PCR-based process with a pair of primers specific for the polymorphic sequence. The association between the polymorphism and schizophrenia was examined in a Japanese sample. There was a statistically significant association between the polymorphism and schizophrenia in females (chi2 = 26.018, p = 0.010), but not in males (chi2 = 15.995, p = 0.305). The genotype heterozygous for the TH9 and TH6 was significantly decreased in female schizophrenics (chi2 = 5.125, p = 0.0236). These results suggest that TH could be considered as a minor gene contributing to the susceptibility of Japanese female schizophrenia.

Detection of double-stranded PCR amplicons at the attomole level electrosprayed from low nanomolar solutions using FT-ICR mass spectrometry.

An 82-base-pair polymerase chain reaction (PCR) product was amplified from the tetranucleotide short tandem repeat locus within the human tyrosine hydroxylase gene. PCR amplification was carried out using 100 ng of human nuclear DNA obtained from an individual who is homozygotic for the 9.3 allele resulting in a 50.5 kDa amplicon. To generate sufficient material for these investigations, several reactions were pooled and subsequently purified and quantified using UV-vis spectrophotometry. A serial dilution was carried out from a 2 microM stock solution providing solution concentrations down to 5 nM. Measurements were made using hexapole accumulation and gated trapping strategies in a 4.7 Telsa Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) which facilitated detection of the amplicon at the attomole level when electrosprayed from a 5 nM solution with a single acquisition! The signal-to-noise ratio was determined to be 8.3 for the spectrum derived from the 5 nM solution using the magnitude-mode mass spectral peak height for the most abundant charge-state. This remarkable sensitivity for large PCR amplicons will dramatically improve the ability of electrospray ionization mass spectrometry to address important genetic questions for low copy number genes or when the amount of initial template is limited; the latter issue is commonly encountered in DNA forensics. Furthermore, these data represents over 2 orders of magnitude decrease in detection limits over other existing ESI-MS reports concerning PCR products, including those conducted using FTICR-MS.

Synergy between 5′ and 3′ flanking regions of the human tyrosine hydroxylase gene ensures specific, high-level expression in neuroblastoma cells

Factors regulating tyrosine hydroxylase (TH) gene transcription are of major importance in the studies of malignant and degenerative diseases of catecholamine-synthesizing tissues. In this study, we used transient transfection of a reporter gene to show that high-level, tissue-specific TH expression was only achieved when the reporter gene was cloned between a 5′ TH promoter sequence (−513–+1), and, a 3′ TH gene flanking sequence (end of exon 14–+976). We also show that TH mRNA expression level is closely linked to the expression level of the proto-oncogene, MYCN in neuroblastoma tumor cell lines. Taken together our data indicate that MYCN may regulate TH expression in neuroblastoma cells, but not through binding to the 5′ or 3′ TH gene flanking sequences used in our experiments.

Multi-site partitioned delivery of human tyrosine hydroxylase gene with phenotypic recovery in Parkinsonian rats.

Parkinson's disease (PD) is a leading candidate for neurological gene therapy, given our increasing knowledge of the functional anatomy of the striatonigral system and the localized nature of the affected cell populations. Here we report that stereotactic introduction of a human tyrosine hydroxylase (TH-2) gene using multi-site partitioned doses resulted in behavioral recovery in 6-OHDA-lesioned rats, with transient 100% recovery observed in some animals. We also show correlation between numbers of TH-immunoreactive cells and loss of apomorphine induced rotation, with a near-linear relationship between TH expression and phenotypic recovery. Furthermore, the data suggest that only a fraction of striatal cells need to be transduced in order to exert phenotypic effects, and therefore TH partitioned gene transfer may have clinical potential in PD.

Preparation of single-stranded PCR products for electrospray ionization mass spectrometry using the DNA repair enzyme lambda exonuclease.

Electrospray ionization mass spectrometry (ESI-MS) has been utilized to obtain accurate mass measurements of intact PCR products; however, single-stranded PCR products are necessary to detect sequence modifications such as base substitutions, additions or deletions. The locations of these modifications can subsequently be determined using additional stages of mass spectrometry. The recombinant enzyme lambda exonuclease selectively digests one strand of a DNA duplex from a 5' phosphorylated end leaving the complementary strand intact. Using this rapid enzymatic step, we were able to produce single-stranded PCR products by digestion of an intact PCR product derived from the Human Tyrosine Hydroxylase (HUMTHO1) gene, which contains a tetrameric repeating motif. The non-template directed 3' adenylation common when using Taq polymerase resulted in three distinct species (blunt-ended, mono-adenylated and di-adenylated), which added complexity to the spectrum of the double-stranded product. The data from the single-stranded products shows that one strand is preferentially adenylated over the other, which cannot be determined from the mass spectrum of the double-stranded PCR product alone. The ESI-FTICR (Fourier transform ion cyclotron resonance) mass spectra of the lambda exonuclease treated PCR products exhibited less than expected signal-to-noise (S/N) ratios. This is attributed to inaccurate concentration calculations due to remaining double-stranded PCR product amplified with unphosphorylated primers, and to matrix effects contributed by the lambda exonuclease reaction buffer. To further test this hypothesis, we investigated and determined the limit of detection to be 0.27 microM using standard curve statistics for single acquisitions of a synthetic 75-mer. The concentrations of the noncoding and coding strands produced by lambda exonuclease digestion were calculated to be 0.29 and 0.37 microM, respectively, taking into account the presence of double-stranded product. The products were electrosprayed from concentrations at the limit of detection requiring the averaging of 5-10 acquisitions to produce a sufficient S/N ratio, indicating that product concentration, base composition and matrix effects play a combined, significant role in detection of lambda exonuclease treated PCR products. Although additional work will be required to further exploit this strategy, lambda exonuclease clearly provides mass spectrometrists with a method to generate single-stranded PCR products.

Dopa-producing astrocytes generated by adenoviral transduction of human tyrosine hydroxylase gene: in vitro study and transplantation to hemiparkinsonian model rats.

Astrocytes secreting a large amount of 3,4-dihydroxyphenylalanine (dopa) were generated by adenoviral transduction of the human tyrosine hydroxylase (TH) gene. After characterizing in vitro, the effect of transplantation of these astrocytes to the striatum of hemiparkinsonian model rats was investigated. Subconfluent cortical astrocytes were infected by replication-defect adenovirus type 5 carrying the human TH-1 gene or the LacZ reporter gene under the promoter of the glial fibrillary acidic protein (AdexGFAP-HTH-1, AdexGFAP-NL-LacZ). Dopa secretion was not evident at 3 days after the transduction of the HTH-1 gene but it increased from 7 days up to at least 4 months. The secretion was substrate (tyrosine)-dependent, and was enhanced by loading tetrahydrobioputerin (BH4) concentration-dependently. One-third of the hemiparkinsonian model rats, that were transplanted the HTH-1 gene-transduced astrocytes or introduced the direct injection of the viral vector to the striatum, showed a reduction of methamphetamine-induced rotations for at least 6 weeks. Apomorphine-induced rotation was decreased to the 50% level of the control's, but the reduction was obtained equally by the transplantation of HTH-1 gene-transduced or LacZ reporter gene-transduced astrocytes, or by the introduction of HTH-1 or LacZ gene carrying adenovirus. Treatment with FK506 for 3 weeks improved the late-phase apomorphine-induced rotations following the introduction of the HTH-1 gene carrying adenovirus. Histological examination revealed that, in animals that showed a reduction of methamphetamine-rotation, the TH positive astrocytes-like cells were distributed widely in the host striatum for at least 4 weeks. The number of TH positive astrocytes-like cells and their immunoreactivity decreased after 6 weeks when OX-41 positive microglias/macrophages were infiltrated. Data indicate that the adenoviral transduction of the human TH gene to astrocytes and its introduction to the striatum is a promising approach for the treatment of Parkinson's disease. However, the further technical improvements are required to optimize the adenoviral gene delivery, such as the control of viral toxicity and the regulation of the immune response.

CDKN2 Mutation Is Infrequent in German Hepatocellular Carcinoma

For hepatocellular carcinoma, only scarce and controversial data on CDKN2 alterations are available. A high rate of mutations in a Chinese study contrasts with a low rate found in Japanese tumors and a CDKN2 germline mutation in 4/26 Swiss tumors examined. We analyzed 23 hepatocellular carcinomas from German patients for homozygous deletions of CDKN2 by coamplification with the human tyrosine hydroxylase (TH) gene and for CDKN2 mutations by PCR-single strand conformation polymorphism analysis and direct DNA sequencing. Our results indicate the lack of homozygous deletions. In one tumor, DNA sequencing showed a GCG-ACG (alanine-threonine) substitution at codon 148, a polymorphism in exon 2 of CDKN2. We conclude that the alteration of CDKN2 by deletion or mutation appears not to be a frequent event in hepatocarcinogenesis in German patients.

A review of biochemical and molecular genetic aspects of tyrosine hydroxylase deficiency including a novel mutation (291delC).

An overview is given of the current knowledge on the human tyrosine hydroxylase gene and on the biochemical aspects of diagnosing defects in this gene. Diagnostic biochemical findings are described in four cases of genetically confirmed tyrosine hydroxylase deficiency. Decreased CSF levels of homovanillic acid (HVA) and 3-methoxy-4-hydroxyphenylethyleneglycol (MHPG), together with normal pterin and CSF tyrosine and 5-hydroxyindoleacetic acid (5-HIAA) concentrations are the diagnostic hallmarks of tyrosine hydroxylase deficiency. At the metabolite level the diagnosis can only be made reliably in CSF. Strict adherence to a standardized lumbar puncture protocol and adequate reference values are essential for diagnosis of this 'new' treatable neurometabolic disorder. Measurements of HVA, vanillylmandelic acid (VMA) or catecholamines in urine are not relevant for diagnosing tyrosine hydroxylase deficiency. The diagnosis should be considered in all children with unexplained hypokinesia and other extrapyramidal symptoms. Three of our patients are homozygous for a mutation in exon 6 (698G > A) of the tyrosine hydroxylase gene and one patient was compound heterozygous for the same mutation and a novel truncating mutation in exon 3 (291delC).

Accurate characterization of the tyrosine hydroxylase forensic allele 9.3 through development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Accurate and precise determination of the number of repeats from a short tandem repeat (STR) sequence for a human gene locus is demonstrated for the first time by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS). Specifically, the polymorphic human tyrosine hydroxylase (HUMTHO1) gene, a tetranucleotide STR forensic allele, was chosen as a model system to evaluate our approach for future characterization of both STRs and variable number of tandem repeats (VNTRs) by development of an ESI-FTICR-MS approach. The coding and noncoding strands from the HUMTHO1 9.3 allele are simultaneously resolved obtaining accurate (better than 70 ppm) average mass measurements of 25,783.23 and 24,754.55 Da for the coding and noncoding strands, respectively. The mass measurements are used to calculate the number of repeats for each strand, 'n', of 9.75169 and 9.75001 for the coding and noncoding strands, respectively. It will be shown how the value of 'n' can be used to directly determine the number of pure repeats and accurately determine the exact nature of the polymorphism within the repeat (if any). The single nucleotide deletion in the coding strand (adenine) and noncoding strand (thymine) were accurately identified using this approach. Interestingly, we observed the conversion of single-stranded to double-stranded DNA while the PCR product in the ESI buffer was being infused; the issues related to this observation will be presented. Previous results by other researchers investigating the HUMTHO1 9.3 allele using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) are directly compared with our results. Our results indicate that ESI-FTICR-MS is a powerful approach to rapidly and accurately characterize tandem repeating sequences which will ultimately lead towards the understanding of a complex class of diseases and in human identity determination.

The Short Tandem Repeat Loci hTPO, THO1 and FGA

This paper focuses on the three well-established short tandem repeats HumhTPO (human thyroid peroxidase gene), HumTHO1 (human tyrosine hydroxylase gene) and HumFGA (human alpha fibrinogen gene). We present sequencing data which include the sequences of all the different primer pairs used so far, as well as results from a population study which was carried out on an American Black and a southwestern German population. All data are compared with previously published studies. A test for homogeneity confirmed that non-European populations possess different genetic pools for each of the three loci under investigation.

No association between the tyrosine hydroxylase microsatellite marker HUMTH01 and schizophrenia or bipolar I disorder.

Linkage and association studies have implicated the involvement of the tyrosine hydroxylase (TH) gene on chromosome 11p15 in schizophrenia and bipolar disorder (BPD). An association of BPD with a polymorphic tetranucleotide repeat, HUMTH01, located in the first intron of the human TH gene has been reported. Subsequently a rare allele, Ep ([TCAT]10) of this microsatellite marker has been found in French and Tunisian schizophrenic patients only. We have genotyped a different sample of unrelated French schizophrenic and BPD patients from Alsace and matched controls for this polymorphic tetranucleotide repeat sequence. The Ep allele was insignificantly more common in controls than in schizophrenic patients, thus not showing a particular association with schizophrenia. In addition, analysis of the segregation of the Ep allele in the family of one of the schizophrenic patients showed no transmission of this allele from the healthy mother to her schizophrenic daughter. Nevertheless, we did observe a non-significant trend towards an association between HUMTH01 allele D ([TCAT]9) and schizophrenia (Fisher's exact test, p = 0.053). No association was apparent between HUMTH01 and BPD Psychiatr Genet.

Possible association of catecholamine turnover with the polymorphic (TCAT) N repeat in the first intron of the human tyrosine hydroxylase gene

Five allelic fragments were typed by a PCR-based process with a pair of primers specific for the polymorphic sequence due to (TCAT)n tetranucleotide repeat, a microsatellite repeat, in the first intron of the human tyrosine hydroxylase gene, i.e. A1, A2, A3, A4 and A5. Comparisons of some neurochemical parameters of the catecholamine pathway were then made between the unrelated individuals genotypically classified into six subgroups, five homozygotic and one heterozygotic. Among the six subgroups, the individuals with the A2 A2 genotype had the highest levels of serum noradrenaline and those with the A4 A4 genotype had the lowest, and the individuals with the A1 A1 genotype had the highest levels of serum homovanillic acid. These findings suggest that polymorphism of the (TCAT)n repeat in the first intron of the human tyrosine hydroxylase gene may be associated with catecholamine turnover. Possibly, the two alleles, A2 and A4, may be related to the high and low excitabilities of the noradrenergic nerves, respectively, and the allele, A1, may be associated with the up-regulation of dopamine turnover.

Generation of DOPA-Producing Astrocytes by Retroviral Transduction of the Human Tyrosine Hydroxylase Gene:In VitroCharacterization andin VivoEffects in the Rat Parkinson Model

Astrocytes secreting high levels ofl-3,4-dihydroxyphenylalanine (DOPA) have been generated by retrovirus-mediated transfer of the human tyrosine hydroxylase (TH) gene. Immature astrocytes obtained from prenatal rat brain were cocultured with TH virus producing Ψ-2 cells that had been pretreated with the mitosis inhibitor mitomycin-C. During the first week of coculture DOPA production gradually increased to reach a plateau after 7–9 days. At this time point virtually all cells were GFAP positive and over 80% of them expressed TH. DOPA production in the transduced astrocytes was largely independent of exogenous cofactor, and DOPA release into the medium was not influenced by addition of either KCl or tetrodotoxin or by removal of Ca2+from the culture medium, indicating that the newly synthesized DOPA was constitutively released from the cells. Transplantation of the TH-transduced astrocytes to the striatum in unilaterally 6-hydroxydopamine lesioned rats reduced apomorphine-induced turning by about 50% at 2 weeks postgrafting. Microscopic analysis revealed that the transduced astrocytes survived very well after transplantation and that some of the grafted cells had migrated out, partly along blood vessels, into the surrounding striatum. TH expression was observed in cells with both the appearance of mature GFAP-positive astrocytes, as well as in more immature-looking cells. However, only a few percent of all transplanted cells maintained significant expression of the transgene, as determined by TH immunohistochemistry. The results show that primary astrocytes may be highly useful as gene carriers forex vivogene therapy in the CNS. With future improvement in the gene transduction procedure for more efficient, sustained expression of the TH transgenein vivo,genetically engineered DOPA-producing astrocytes hold great promise as a tool to explore the potential ofex vivogene therapy in Parkinson's disease.

Association of polymorphic microsatellite region in the first intron of the human tyrosine hydroxylase gene with disturbances of the catecholaminergic system in schizophrenia

Association studies with the DRD2 Taq1A marker have been variable in implicating DRD2 as a “Reward Deficiency Syndrome Gene” for alcoholism and substance abuse. Given that the Taq1A marker is not functionally significant, second-generation studies on the DRD2 receptor to identify functional variants and evaluate their effect on the phenotype are the logical step towards confirming and extending the DRD2 hypothesis. This article discusses the implications and process of progress made in these directions. The new findings are the description of structural variants in the D2 receptor, the demonstration that one of these, Ser311Cys, largely prevents signal transduction following receptor activation and the use of Ser311Cys in a large association and sib-pair linkage anlysis in an American Indian isolate. In this particular population, the Cys311 variant is far more abundant (0.16) than in Caucasians (0.03). Genotyping of Ser311Cys, the DRD2 intron 2 STR, and the Taq1A marker in 459 subjects, including 373 sib-pairs and 15 Cys311/Cys311 homozygous individuals, revealed no association to alcoholism, substance use disorders, or schizophrenia. The implication is that a DRD2 variant that dramatically impairs receptor function was not sufficient to significantly alter alcoholism vulnerability in a relatively large and also genetically and environmentally homogeneous sample. Published by Elsevier Science Inc.

The 3' flanking region of the human tyrosine hydroxylase gene directs reporter gene expression in peripheral neuroendocrine tissues.

Cell type-specific expression of the catecholamine synthetic enzyme, tyrosine hydroxylase (TH), appears to be mediated in part by cis-acting elements located at the 3' end of the human gene. Further delineation of this region indicated sequences corresponding to a CACGTG motif significantly stimulated transcription of a heterologous promoter in various cell types. Mutation of this site led to a complete loss of activity. DNase footprinting, gel retardation, and UV cross-linking experiments indicated that a 74-kDa cellular factor(s) bound specifically to the CACGTG motif in the pheochromocytoma cell line PC12. The size of this protein and its pattern of expression are compatible with those of the CACGTG binding protein TFE3. Transgenic animals were created using a 261-bp human TH 3' fragment encompassing the CACGTG motif in front of a thymidine kinase promoter/chloramphenicol acetyltransferase reporter gene. In three lines of mice this fragment was sufficient to direct a pattern of mRNA expression in peripheral neuroendocrine tissues that mimicked TH mRNA distribution. However, these sequences were not sufficient for CNS-specific patterns of expression. Thus, multiple cell type-specific enhancers may regulate TH gene expression in the CNS and periphery.

Frequent sequence variant in the human tyrosine hydroxylase gene

A polymorphism of human tyrosine hydroxylase changing the amino acid 81Val to 81Met is located in exon 2 of the human tyrosine hydroxylase gene.

Association of polymorphic VNTR region in the first intron of the human TH gene with disturbances of the catecholamine pathway in schizophrenia.

In the present study, five allelic fragments were typed by a polymerase chain reaction (PCR) process with a pair of primers specific for the tetranucleotide (TCAT) repeat sequence in the first intron of the human tyrosine hydroxylase (TH) gene and their sizes (bp) were 114 (A), 118 (B), 122 (C), 126 (D) and 130 (E), respectively. The AE genotypic frequency was found to be significantly higher in unrelated patients with schizophrenia than in unrelated control subjects (chi 2 = 4.18, p < 0.05). ANOVA revealed a significant difference between the three groups (neuroleptic-free patients possessing or not possessing the AE genotype, and unrelated control subjects) in the concentration of serum noradrenaline (F = 4.96, df = 2.79, p < 0.01), but no significant differences were found between the three groups in the concentrations of serum homovanillic acid, phenylalanine and tyrosine. These results suggest that the polymorphic intron 1 of the human TH gene may be associated with disturbances of the catecholamine pathway in schizophrenia.

Peripherally administered (6 R)-tetrahydrobiopterin increases in vivo tyrosine hydroxylase activity in the striatum measured by microdialysis both in normal mice and in transgenic mice carrying human tyrosine hydroxylase

The intraperitoneal administration of (6 R)- L- erythro-5,6,7,8-tetrahydrobiopterin (6 R-BH4), the natural cofactor of tyrosine hydroxylase (TH), increased the accumulation of l-3,4-dihydroxyphenylalanine (DOPA) measured using microdialysis under the inhibition of aromatic l-amino acid decarboxylase by NSD-1015 (in vivo TH activity) in the striatum both of transgenic mice carrying human TH gene and of non-transgenic mice, to a similar extent by about 4-fold. The results indicate that the peripherally administered 6 R-BH4 activates in vivo TH activity in the nigrostriatal dopamine neurons in both non-transgenic and transgenic mice.

A new highly polymorphic DNA restriction site marker in the 5' region of the human tyrosine hydroxylase gene (TH) detecting loss of heterozygosity in human embryonal rhabdomyosarcoma.

We have isolated a new marker (cos11-5TH) that detects an MspI restriction fragment length polymorphism in the 5' region of the human tyrosine hydroxylase gene (TH) on chromosome band 11p15.5. This region of human chromosome 11 contains several important loci for disease phenotypes including Beckwith-Wiedemann syndrome (BWS), Wilms' tumor, and embryonal rhabdomyosarcoma. Thus, identification of new polymorphic markers in this region are important for future gene mapping and linkage analyses. To better define the region of 11p15.5 deleted in embryonal rhabdomyosarcoma, this new marker was used to investigate allelic losses in embryonal rhabdomyosarcoma tumors.