Virological analysis of inherited chromosomally integrated human herpesvirus-6 in three hematopoietic stem cell transplant patients.

We analyzed 3 hematopoietic stem cell transplant (HSCT) recipients with inherited chromosomally integrated human herpesvirus-6 (inherited CIHHV-6). Cases 1 (inherited CIHHV-6A) and 2 (inherited CIHHV-6B) were inherited CIHHV-6 recipients. Case 3 received bone marrow from a donor with inherited CIHHV-6B. Following HSCT, HHV-6B was isolated from Case 1. HHV-6A and -6B messenger RNAs were detected in Cases 1 and 3.

The constitutional t(11;22): implications for a novel mechanism responsible for gross chromosomal rearrangements.

The constitutional t(11;22)(q23;q11) is the most common recurrent non-Robertsonian translocation in humans. The breakpoint sequences of both chromosomes are characterized by several hundred base pairs of palindromic AT-rich repeats (PATRRs). Similar PATRRs have also been identified at the breakpoints of other nonrecurrent translocations, suggesting that PATRR-mediated chromosomal translocation represents one of the universal pathways for gross chromosomal rearrangement in the human genome. We propose that PATRRs have the potential to form cruciform structures through intrastrand-base pairing in single-stranded DNA, creating a source of genomic instability and leading to translocations. Indeed, de novo examples of the t(11;22) are detected at a high frequency in sperm from normal healthy males. This review synthesizes recent data illustrating a novel paradigm for an apparent spermatogenesis-specific translocation mechanism. This observation has important implications pertaining to the predominantly paternal origin of de novo gross chromosomal rearrangements in humans.

A new splicing variant for human tyrosine hydroxylase in the adrenal medulla.

It has been reported that several mRNA isoforms of tyrosine 3-monooxygenase (tyrosine hydroxylase; TH) occur only in primates. New TH isoforms produced by skipping of exon 3 in the adrenal medulla of patients with progressive supranuclear palsy (PSP) have recently been reported, J. Neurochem. 67 (1996) 19. Here, we looked for the presence of new TH isoforms in control brains and adrenal medulla and in brains from patients with PSP. We found a novel type of TH mRNA in the adrenal medulla from one of the control subjects. The mRNA lacked exon 4, resulting in a premature stop codon at amino acid 147. This result suggests the importance of alternative splicing in the regulation of TH activity.

Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice.

(6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase (TH), tryptophan hydroxylase, phenylalanine hydroxylase, and nitric-oxide synthase. These enzymes synthesize neurotransmitters, e.g. catecholamines, serotonin, and nitric oxide (NO). We established mice unable to synthesize BH4 by disruption of the 6-pyruvoyltetrahydropterin synthase gene, the encoded protein of which catalyzes the second step of BH4 biosynthesis. Homozygous mice were born at the almost expected Mendelian ratio, but died within 48 h after birth. In the brain of homozygous mutant neonates, levels of biopterin, catecholamines, and serotonin were extremely low. The number of TH molecules was highly dependent on the intracellular concentration of BH4 at nerve terminals. Alteration of the TH protein level by modulation of the BH4 content is a novel regulatory mechanism. Our data showing that catecholaminergic, serotonergic, and NO systems were differently affected by BH4 starvation suggest the possible involvement of BH4 synthesis in the etiology of monoamine-based neurological and neuropsychiatric disorders.

Quantification of tyrosine hydroxylase mRNA.

The main biochemical characteristic of Parkinson's disease (PD) is reduction of the neurotransmitter dopamine and the dopamine-synthesizing enzyme system, including tyrosine hydroxylase (TH, tyrosine 3-monooxygenase, EC 1.14.16.2) and tetrahydrobiopterin (BH(4) co-factor, in nigrostriatal neurons (1). The deficiency in dopamine-synthesizing enzymes is accompanied by cell loss, which is thought to be caused by unknown exogenous environmental factors as well as endogenous genetic factors.

Molecular mechanisms of hereditary progressive dystonia with marked diurnal fluctuation, Segawa's disease.

The causative gene for hereditary progressive dystonia with marked diurnal fluctuation/dopa-responsive dystonia (HPD/DRD) was discovered in 1994 to be guanosine triphosphate (GTP) cyclohydrolase I, an enzyme involved in tetrahydrobiopterin biosynthesis. To the present, more than 50 mutations have been found in this gene in HPD/DRD patients. Although it is clear that HPD/DRD is caused by partial deficiency of tetrahydrobiopterin in the brain, several important issues regarding the molecular etiology of HPD/DRD remain to be addressed. We review herein the recent progress in the molecular genetics of HPD/DRD and clarify the points to be answered.

Characterization of wild-type and mutants of recombinant human GTP cyclohydrolase I: relationship to etiology of dopa-responsive dystonia.

To explore the molecular etiology of two disorders caused by a defect in GTP cyclohydrolase I--hereditary progressive dystonia with marked diurnal fluctuation (HPD), also known as dopa-responsive dystonia (DRD), and autosomal recessive GTP cyclohydrolase I deficiency--we purified and analyzed recombinant human wild-type and mutant GTP cyclohydrolase I proteins expressed in Escherichia coli. Mutant proteins showed very low enzyme activities, and some mutants were eluted at a delayed volume on gel filtration compared with the recombinant wild-type. Next, we examined the GTP cyclohydrolase I protein amount by western blot analysis in phytohemagglutinin-stimulated mononuclear blood cells from HPD/DRD patients. We found a great reduction in the amount of the enzyme protein not only in one patient who had a frameshift mutation, but also in an HPD/DRD patient who had a missense mutation. These results suggest that a dominant-negative effect of chimeric protein composed of wild-type and mutant subunits is unlikely as a cause of the reduced enzyme activity in HPD/DRD patients. We suggest that reduction of the amount of the enzyme protein, which is independent of the mutation type, could be a reason for the dominant inheritance in HPD/DRD.

[The relation between metabolism of biopterin and dystonia-parkinsonism].

Tetrahydrobiopterin (BH4) is an essential cofactor for tyrosine hydroxylase. BH4 can be synthesized from GTP through three enzymatic reactions. The rate-limiting step of the BH4 synthesis is catalyzed by GTP cyclohydrolase I (GCH). Recently, we found that GCH is a causative gene for hereditary progressive dystonia/dopa-responsive dystonia (HPD/DRD). However, several problems still remain to be solved. The first concern is the presence of asymptomatic carriers in the disease. The difference between symptomatic and asymptomatic carriers is unknown. Second, we cannot find any mutation in the coding region of the GCH gene in about 40% of the patients. What kind of mutation would be present in these patients. The last concern is the molecular mechanism how the enzymatic activity is decreased to less than 20% of normal values. Further studies are required to solve the questions.

Decrease in GTP cyclohydrolase I gene expression caused by inactivation of one allele in hereditary progressive dystonia with marked diurnal fluctuation.

Hereditary progressive dystonia with marked diurnal fluctuation (HPD; dopa-responsive dystonia, DRD) have been recently found to be caused by a genetic defect in the GTP cyclohydrolase I (GCH1) gene. In this study, we quantified the mRNA level of GCH1 in phytohemagglutinin (PHA)-stimulated mononuclear blood cells from one Japanese family that do not have a mutation in the coding region or splice junctions of the gene. The results showed that the amounts of the GCH1 mRNA were decreased to about 40% of the normal level in both patients and carriers. In addition, we found that the GCH1 mRNA was transcribed from only one allele, indicating that the other allele was in an inactive state. These results suggest that some novel mutations should exist on one of the alleles in some unknown region of the GCH1 gene, and may decrease the GCH1 mRNA causing the HPD/DRD symptoms.

Molecular cloning of the human Nurr1 gene: characterization of the human gene and cDNAs.

Nurr1 is a member of the nuclear receptor superfamily of transcription factors that is expressed predominantly in the central nervous system, including developing dopaminergic neurons. Recently, it was demonstrated that Nurr1 is critical for midbrain dopaminergic cell differentiation. In order to investigate a possible relation of Nurr1 with the pathogenesis of Parkinson's disease or other neuropsychiatric disorders, we have cloned and characterized the human Nurr1 gene. The gene exists as a single copy in the human genome and comprises eight exons spanning 8kb. We determined the complete nucleotide sequence and flanking regions of the gene. Potential regulatory regions included consensus binding sites for NF-kappaB, CREB, and Sp1. Isolation of human Nurr1 cDNAs from fetal brain suggested the presence of a new splicing variant of Nurr1 in the human brain.

Molecular genetics of dopa-responsive dystonia.

The causative genes of two types of hereditary dopa-responsive dystonia (DRD) due to dopamine (DA) deficiency in the nigrostriatum DA neurons have been elucidated. Autosomal dominant DRD (AD-DRD) was originally described by Segawa as hereditary progressive dystonia with marked diurnal fluctuation (HPD). We cloned the human GTP cyclohydrolase I (GCH1) gene, and mapped the gene to chromosome 14q22.1-q22.2 within the HPD/DRD locus, which had been identified by linkage analysis. GCH1 isthe rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), the cofactor for tyrosine hydroxylase (TH), which is the first and rate-limiting enzyme of DA synthesis. We proved that the GCH1 gene is the causative gene for HPD/DRD based on the identification of mutations of the gene in the patients and decreases in the enzyme activity expressed in mononuclear blood cells to 2-20% of the normal value. About 60 different mutations (missense, nonsense, and frameshift mutations) in the coding region or in the exon-intron junctions of the GCH1 gene have been reported in patients with AD-DRD all over the world. Recent findings indicate that the decreased GCH1 activity in AD-DRD may be caused by the negative interaction of the mutated subunit with the wild-type one, i.e., a dominant negative effect, and/or by decreases in the levels of GCH1 mRNA and protein caused by inactivation of one allele of the GCH1 gene. Autosomal recessive DRD (AR-DRD) with Segawa's syndrome was discovered in Germany. The AR-DRD locus was mapped to chromosome 11p15.5 in the chromosomal site of the TH gene. In the AR-DRD with Segawa's syndrome, a point mutation in TH (Gln381Lys) resulted in a pronounced decrease in TH activity to about 15% of that of the wild type. Several missense mutations in the TH gene have been found in AR-DRD in Europe. The phenotype of AR-DRD with the Leu205Pro mutation in the TH gene, which produces a severe decrease in TH activity to 1.5% of that of the wild type, was severe, not dystonia/Segawa's syndrome, but early-onset parkinsonism. However, a marked improvement of all clinical symptoms with a low dose of L-dopa was reported in AR-DRD/parkinsonism patients. These findings on DRD indicate that the nigrostriatal DA neurons may be most susceptible to the decreases in GCH1 activity, BH4 level, TH activity, and DA level, and that DRD is the DA deficiency without neuronal death in contrast to juvenile parkinsonism or Parkinson's disease with DA cell death.

Genomic organization and chromosomal localization of the human sepiapterin reductase gene.

Sepiapterin reductase (SPR) catalyzes the final step of the biosynthetic pathway of tetrahydrobiopterin, which is an essential cofactor for aromatic amino acid hydroxylases and nitric oxide synthases. To aid the analysis of any possible human diseases caused by mutations in SPR, we have cloned and characterized the human SPR gene. The gene is composed of three exons spanning approximately 4 kilobases. The transcriptional starting point was determined around the cytosine nucleotide at position -81 by primer extension and RT-PCR analyses. There was no typical TATA-box within 300 bp from the transcriptional starting point. We found the Sp1-binding consensus sequence in the 5'-flanking region. The human SPR gene was mapped to chromosome band 2p13 by fluorescence in situ hybridization.

A novel strategy for the negative selection in mouse embryonic stem cells operated with immunotoxin-mediated cell targeting.

Immunotoxoin-mediated cell targeting (IMCT) is a technique for conditionally ablating specific cell types based on the cytotoxic activity of a recombinant immunotoxin anti-Tac (Fv)-PE40. To examine the feasibility of this technique for the negative selection in mouse embryonic stem (ES) cells, we investigated the responsiveness of cells expressing human interleukin-2 receptor alpha subunit to anti-Tac(Fv)-PE40. The immunotoxin treatment efficiently eliminated only ES cells bearing the receptor as a consequence of the target specificity of anti-Tac(Fv)-PE40, indicating that IMCT can be used as a novel strategy for positive and negative selection to enrich ES cell clones with a targeted mutation.

GTP cyclohydrolase I gene in hereditary progressive dystonia with marked diurnal fluctuation.

We previously reported four different mutations in the coding region of GTP cyclohydrolase I (GCH-I) gene in patients with hereditary progressive dystonia with marked diurnal fluctuation (HPD). We found two independent new mutations (leucine 79 proline and a deletion in exon 4) in patients with HPD. We also found four families of HPD without any mutations in the coding region of GCH-I gene.

GTP cyclohydrolase I activity in mononuclear blood cells in juvenile parkinsonism.

GTP cyclohydrolase I activity in mononuclear blood cells from patients with juvenile parkinsonism (JP) was found to be normal compared to healthy controls. The normal activity in JP contrasts strongly with the decreased activity of 2-20% normal levels in hereditary progressive dystonia with marked diurnal fluctuation (HPD) or dopa responsive dystonia (DRD). The result indicates that the decreased dopamine level in the basal ganglia in JP is not due to decreased activity of GTP cyclohydrolase I, the enzyme for the biosynthesis of the tetrahydrobiopterin cofactor of tyrosine hydroxylase (TH), and the enzyme activity in mononuclear blood cells could be a reliable method for differential diagnosis between JP and HPD/DRD.

Characterization of mouse and human GTP cyclohydrolase I genes. Mutations in patients with GTP cyclohydrolase I deficiency.

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 approximately 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.

Alterations in multiple tyrosine hydroxylase mRNAs in the substantia nigra, locus coeruleus and adrenal gland of MPTP-treated parkinsonian monkeys.

Monkeys are known to be highly susceptible to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which produces parkinsonism, as in humans. We have previously reported that only monkeys and humans have multiple isoforms of tyrosine hydroxylase (TH), the first enzyme for dopamine biosynthesis, with only two TH mRNA isoforms, type 1 and type 2, being present in Macaca fascicularis. In the present study we have measured TH mRNA type 1 and 2 content in the substantia nigra, locus coeruleus, and adrenal gland of normal control monkeys and in MPTP-produced parkinsonian monkeys (Macaca fascicularis) using a newly developed, sensitive and quantitative assay based on the reverse transcription-polymerase chain reaction. Marked decreases in TH mRNA type 1 and 2 content were observed, specifically in the substantia nigra of the monkeys with MPTP-parkinsonism compared to control monkeys. These results are similar to our recent data showing marked decreases in TH mRNA type 1, 2, 3 and 4 content in the substantia nigra of patients with Parkinson's disease, and suggest that MPTP-treated monkeys closely replicate changes in TH isoforms in human Parkinson's disease.

Hereditary progressive dystonia with marked diurnal fluctuation caused by mutations in the GTP cyclohydrolase I gene.

Hereditary progressive dystonia with marked diurnal fluctuation (HPD) (also known as dopa responsive dystonia) is a dystonia with onset in childhood that shows a marked response without any side effects to levodopa. Recently the gene for dopa responsive dystonia (DRD) was mapped to chromosome 14q. Here we report that GTP cyclohydrolase I is mapped to 14q22.1-q22.2. The identification of four independent mutations of the gene for GTP cyclohydrolase I in patients with HPD, as well as a marked decrease in the enzyme's activity in mononuclear blood cells, confirms that the GTP cyclohydrolase I gene is a causative gene for HPD/DRD. This is the first report of a causative gene for the inherited dystonias.