41 results on '"Ohye, T."'
Search Results
2. Characterization of wild-type and mutants of recombinant human GTP cyclohydrolase I: Relationship to etiology of dopa-responsive dystonia
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Suzuki, T., Ohye, T., Inagaki, H., Nagatsu, T., and Ichinose, Hiroshi
- Subjects
medicine.medical_specialty ,Phenylalanine ,Recombinant Fusion Proteins ,GTP cyclohydrolase I ,DNA Mutational Analysis ,Molecular Sequence Data ,Mutant ,Genes, Recessive ,Biology ,Biochemistry ,Frameshift mutation ,Neuroblastoma ,Cellular and Molecular Neuroscience ,Internal medicine ,Tumor Cells, Cultured ,medicine ,Humans ,Point Mutation ,Missense mutation ,Amino Acid Sequence ,Frameshift Mutation ,GTP Cyclohydrolase ,Genes, Dominant ,Wild type ,Tetrahydrobiopterin ,Biopterin ,Fusion protein ,Endocrinology ,Gene Expression Regulation ,Dystonic Disorders ,biology.protein ,Dystonic disorder ,medicine.drug - Abstract
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.
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- 2002
3. Molecular cloning of the human Nurr1 gene: characterization of the human gene and cDNAs
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Ichinose, Hiroshi, Ohye, T., Suzuki, T., Sumi-Ichinose, C., Nomura, T., Hagino, Y., and Nagatsu, T.
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DNA, Complementary ,Molecular Sequence Data ,Restriction Mapping ,Nerve Tissue Proteins ,Biology ,Exon ,Sequence Homology, Nucleic Acid ,Nuclear Receptor Subfamily 4, Group A, Member 2 ,Genetics ,Humans ,Cloning, Molecular ,Gene ,Binding Sites ,Base Sequence ,Alternative splicing ,Intron ,Brain ,Parkinson Disease ,General Medicine ,Exons ,HNF1B ,Introns ,DNA-Binding Proteins ,Alternative Splicing ,Regulatory sequence ,RNA splicing ,Schizophrenia ,Human genome ,Transcription Factors - Abstract
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.
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- 1999
4. Polymorphisms of the 22q11.2 breakpoint region influence the frequency of de novo constitutional t(11;22)s in sperm
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Tong, M., primary, Kato, T., additional, Yamada, K., additional, Inagaki, H., additional, Kogo, H., additional, Ohye, T., additional, Tsutsumi, M., additional, Wang, J., additional, Emanuel, B. S., additional, and Kurahashi, H., additional
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- 2010
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5. Two different forms of palindrome resolution in the human genome: deletion or translocation
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Kato, T., primary, Inagaki, H., additional, Kogo, H., additional, Ohye, T., additional, Yamada, K., additional, Emanuel, B. S., additional, and Kurahashi, H., additional
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- 2008
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6. Cruciform extrusion propensity of human translocation-mediating palindromic AT-rich repeats
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Kogo, H., primary, Inagaki, H., additional, Ohye, T., additional, Kato, T., additional, Emanuel, B. S., additional, and Kurahashi, H., additional
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- 2007
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7. Characterization of mouse and human GTP cyclohydrolase I genes. Mutations in patients with GTP cyclohydrolase I deficiency.
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Ichinose, H, Ohye, T, Matsuda, Y, Hori, T, Blau, N, Burlina, A, Rouse, B, Matalon, R, Fujita, K, and Nagatsu, T
- 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 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.
- Published
- 1995
8. Target enrichment long-read sequencing with adaptive sampling can determine the structure of the small supernumerary marker chromosomes.
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Mariya T, Kato T, Sugimoto T, Miyai S, Inagaki H, Ohye T, Sugihara E, Muramatsu Y, Mizuno S, and Kurahashi H
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- Genetic Markers, Humans, In Situ Hybridization, Fluorescence, Microarray Analysis, Chromosomes, Mosaicism
- Abstract
Structural analysis of small supernumerary marker chromosomes (sSMCs) has revealed that many have complex structures. Structural analysis of sSMCs by whole genome sequencing using short-read sequencers is challenging however because most present with a low level of mosaicism and consist of a small region of the involved chromosome. In this present study, we applied adaptive sampling using nanopore long-read sequencing technology to enrich the target region and thereby attempted to determine the structure of two sSMCs with complex structural rearrangements previously revealed by cytogenetic microarray. In adaptive sampling, simple specification of the target region in the FASTA file enables to identify whether or not the sequencing DNA is included in the target, thus promoting efficient long-read sequencing. To evaluate the target enrichment efficiency, we performed conventional pair-end short-read sequencing in parallel. Sequencing with adaptive sampling achieved a target enrichment at about a 11.0- to 11.5-fold higher coverage rate than conventional pair-end sequencing. This enabled us to quickly identify all breakpoint junctions and determine the exact sSMC structure as a ring chromosome. In addition to the microhomology and microinsertion at the junctions, we identified inverted repeat structure in both sSMCs, suggesting the common generation mechanism involving replication impairment. Adaptive sampling is thus an easy and beneficial method of determining the structures of complex chromosomal rearrangements., (© 2021. The Author(s), under exclusive licence to The Japan Society of Human Genetics.)
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- 2022
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9. Andrographolide, isolated from Andrographis paniculata , induces apoptosis in monocytic leukemia and multiple myeloma cells via augmentation of reactive oxygen species production.
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Doi H, Matsui T, Dijkstra JM, Ogasawara A, Higashimoto Y, Imamura S, Ohye T, Takematsu H, Katsuda I, and Akiyama H
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- Andrographis paniculata, Apoptosis, Cell Line, Tumor, Cytarabine pharmacology, Humans, Reactive Oxygen Species metabolism, Diterpenes pharmacology, Hematologic Neoplasms, Leukemia, Multiple Myeloma drug therapy
- Abstract
Background : Andrographolide (Andro) is a diterpenoid component of the plant Andrographis paniculata that is known for its anti-tumor activity against a variety of cancer cells. Methods : We studied the effects of Andro on the viability of the human leukemia monocytic cell line THP-1 and the human multiple myeloma cell line H929. Andro was compared with cytosine arabinoside (Ara-C) and vincristine (VCR), which are well-established therapeutics against hematopoietic tumors. The importance of reactive oxygen species (ROS) production for the toxicity of each agent was investigated by using an inhibitor of ROS production, N-acetyl-L-cysteine (NAC). Results : Andro reduced the viability of THP-1 and H929 in a dose-dependent manner. H929 viability was highly susceptible to Andro, although only slightly susceptible to Ara-C. The agents Andro, Ara-C, and VCR each induced apoptosis, as shown by cellular shrinkage, DNA fragmentation, and increases in annexin V-binding, caspase-3/7 activity, ROS production, and mitochondrial membrane depolarization. Whereas Ara-C and VCR increased the percentages of cells in the G0/G1 and G2/M phases, respectively, Andro showed little or no detectable effect on cell cycle progression. The apoptotic activities of Andro were largely suppressed by NAC, an inhibitor of ROS production, whereas NAC hardly affected the apoptotic activities of Ara-C and VCR. Conclusions : Andro induces ROS-dependent apoptosis in monocytic leukemia THP-1 and multiple myeloma H929 cells, underlining its potential as a therapeutic agent for treating hematopoietic tumors. The high toxicity for (thus forming: The high toxicity for H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma.) H929 cells, by a mechanism that is different from that of Ara-C and VCR, is encouraging for further studies on the use of Andro against multiple myeloma., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Doi H et al.)
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- 2021
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10. Coinfection With Human Herpesvirus (HHV)-6B in Immunocompetent, Healthy Individuals With Chromosomally Integrated HHV-6A.
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Miura H, Ohye T, Kozawa K, Hattori F, Kawamura Y, Ihira M, Kurahashi H, and Yoshikawa T
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- Humans, Saliva, Coinfection, Herpesvirus 6, Human genetics, Roseolovirus Infections diagnosis
- Abstract
Immunocompetent sisters with chromosomally integrated human herpesvirus 6A (HHV-6A) transiently excreted HHV-6B genome in their saliva. They did not have past histories of exanthema subitum but had antibodies against HHV-6A and HHV-6B. This suggests that endogenous HHV-6A may modify the clinical features of HHV-6B coinfection., (© The Author(s) 2020. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2021
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11. Tyrosine hydroxylase conditional KO mice reveal peripheral tissue-dependent differences in dopamine biosynthetic pathways.
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Miyajima K, Kawamoto C, Hara S, Mori-Kojima M, Ohye T, Sumi-Ichinose C, Saito N, Sasaoka T, Metzger D, and Ichinose H
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- Animals, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Organ Specificity, Adrenal Glands metabolism, Biosynthetic Pathways, Catecholamines metabolism, Dopamine biosynthesis, Neurons metabolism, Sympathetic Nervous System metabolism, Tyrosine 3-Monooxygenase physiology
- Abstract
Dopamine (DA) exerts well-known functions in the brain as a neurotransmitter. In addition, it plays important physiological roles in peripheral organs, but it is largely unknown how and where peripheral DA is synthesized and regulated. Catecholamines in peripheral tissues are either produced within the tissue itself and/or derived from sympathetic neurons, which release neurotransmitters for uptake by peripheral tissues. To evaluate DA-producing ability of each peripheral tissue, we generated conditional KO mice (cKO mice) in which the tyrosine hydroxylase (TH) gene is ablated in the sympathoadrenal system, thus eliminating sympathetic neurons as a DA source. We then examined the alterations in the noradrenaline (NA), DA, and 3,4-dihydroxyphenylalanine (DOPA) contents in peripheral organs and performed immunohistochemical analyses of TH-expressing cells. In the heart and pancreas of cKO mice, both the TH protein and NA levels were significantly decreased, and the DA contents were decreased in parallel with NA contents, indicating that the DA supply originated from sympathetic neurons. We found TH-immunoreactive cells in the stomach and lung, where the TH protein showed a decreasing trend, but the DA levels were not decreased in cKO mice. Moreover, we found a significant correlation between the DA content in the kidney and the plasma DOPA concentration, suggesting that the kidney takes up DOPA from blood to make DA. The aforementioned data unravel differences in the DA biosynthetic pathway among tissues and support the role of sympathetic neurons as a DA supplier., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2021
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12. Disruption of the Responsible Gene in a Phosphoglucomutase 1 Deficiency Patient by Homozygous Chromosomal Inversion.
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Yokoi K, Nakajima Y, Ohye T, Inagaki H, Wada Y, Fukuda T, Sugie H, Yuasa I, Ito T, and Kurahashi H
- Abstract
Phosphoglucomutase 1 (PGM1) deficiency is a recently defined disease characterized by glycogenosis and a congenital glycosylation disorder caused by recessive mutations in the PGM1 gene. We report a case of a 12-year-old boy with first-cousin parents who was diagnosed with a PGM1 deficiency due to significantly decreased PGM1 activity in his muscle. However, Sanger sequencing revealed no pathogenic mutation in the PGM1 gene in this patient. As this case presented with a cleft palate in addition to hypoglycemia and elevated transaminases and creatine kinase, karyotyping was performed and identified homozygous inv(1)(p31.1p32.3). Based on the chromosomal location of the PGM1 gene at 1p31, we analyzed the breakpoint of the inversion. Fluorescence in situ hybridization (FISH) combined with long PCR analysis revealed that the inversion disrupts the PGM1 gene within intron 1. Since the initiation codon in the PGM1 gene is located within exon 1, we speculated that this inversion inactivates the PGM1 gene and was therefore responsible for the patient's phenotype. When standard molecular testing fails to reveal a mutation despite a positive clinical and biochemical diagnosis, the presence of a gross structural variant that requires karyotypic examination must be considered.
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- 2019
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13. Analysis of the origin of inherited chromosomally integrated human herpesvirus 6 in the Japanese population.
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Kawamura Y, Ohye T, Miura H, Ihira M, Kato Y, Kurahashi H, and Yoshikawa T
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- Adult, Chromosomes, Human, Pair 22 genetics, Female, Genome, Viral, Herpesvirus 6, Human classification, Herpesvirus 6, Human genetics, Herpesvirus 6, Human physiology, Humans, Japan, Male, Repetitive Sequences, Nucleic Acid, Roseolovirus Infections congenital, Roseolovirus Infections genetics, Virus Integration, Chromosomes, Human, Pair 22 virology, Herpesvirus 6, Human isolation & purification, Roseolovirus Infections virology
- Abstract
Integration of the complete human herpesvirus 6 (HHV-6) genome into the telomere of a chromosome has been reported in some individuals (inherited chromosomally integrated HHV-6; iciHHV-6). Since the proportion of iciHHV-6-positive individuals with integration in chromosome 22 is high in Japan, we hypothesized a founder effect. In this study, we sought to elucidate the reason for the high proportion of viral integrations into chromosome 22. We analyzed six cases of iciHHV-6A and two cases of iciHHV-6B, including one iciHHV-6A case with a matched sample from a father and one iciHHV-6B case with a matched sample from a mother. In iciHHV-6A, the same copy numbers of viral telomeric repeat sequences (TRS) and the same five microsatellite markers were detected in both the index case and paternal sample. Moreover, the same five microsatellite markers were demonstrated in four cases and the same copy numbers of viral TRS were demonstrated in two pairs of two cases. The present microsatellite analysis suggested that the viral genomes detected in some iciHHV-6A patients were derived from a common ancestral integration.
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- 2017
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14. Palindrome-Mediated Translocations in Humans: A New Mechanistic Model for Gross Chromosomal Rearrangements.
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Inagaki H, Kato T, Tsutsumi M, Ouchi Y, Ohye T, and Kurahashi H
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Palindromic DNA sequences, which can form secondary structures, are widely distributed in the human genome. Although the nature of the secondary structure-single-stranded "hairpin" or double-stranded "cruciform"-has been extensively investigated in vitro, the existence of such unusual non-B DNA in vivo remains controversial. Here, we review palindrome-mediated gross chromosomal rearrangements possibly induced by non-B DNA in humans. Recent advances in next-generation sequencing have not yet overcome the difficulty of palindromic sequence analysis. However, a dozen palindromic AT-rich repeat (PATRR) sequences have been identified at the breakpoints of recurrent or non-recurrent chromosomal translocations in humans. The breakages always occur at the center of the palindrome. Analyses of polymorphisms within the palindromes indicate that the symmetry and length of the palindrome affect the frequency of the de novo occurrence of these palindrome-mediated translocations, suggesting the involvement of non-B DNA. Indeed, experiments using a plasmid-based model system showed that the formation of non-B DNA is likely the key to palindrome-mediated genomic rearrangements. Some evidence implies a new mechanism that cruciform DNAs may come close together first in nucleus and illegitimately joined. Analysis of PATRR-mediated translocations in humans will provide further understanding of gross chromosomal rearrangements in many organisms.
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- 2016
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15. Intragenic duplication in the PKHD1 gene in autosomal recessive polycystic kidney disease.
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Miyazaki J, Ito M, Nishizawa H, Kato T, Minami Y, Inagaki H, Ohye T, Miyata M, Boda H, Kiriyama Y, Kuroda M, Sekiya T, Kurahashi H, and Fujii T
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- Amniocentesis methods, Exome, Female, Humans, Male, Pregnancy, Sequence Analysis, DNA, Young Adult, Mutation, Polycystic Kidney, Autosomal Recessive diagnosis, Polycystic Kidney, Autosomal Recessive genetics, Receptors, Cell Surface genetics
- Abstract
Background: In the present study, we report on a couple who underwent prenatal genetic diagnosis for autosomal recessive polycystic kidney disease (ARPKD)., Case Presentation: This healthy couple had previously had a healthy boy but had experienced two consecutive neonatal deaths due to respiratory distress resulting from pulmonary hypoplasia caused by oligohydramnios. The woman consulted our facility after she realized she was pregnant again. We promptly performed a carrier test for the PKHD1 gene by target exome sequencing of samples from the couple. A pathogenic mutation was identified only in the paternal allele (c.9008C>T, p.S3003F). The mutation was confirmed by Sanger sequencing of the DNA from formalin-fixed, paraffin-embedded, kidney tissue of the second neonate patient and was not found in the healthy sibling. We then performed haplotype analyses using microsatellite markers scattered throughout the PKHD1 gene. DNA from the amniocentesis was determined to belong to a carrier, and the couple decided to continue with the pregnancy, obtaining a healthy newborn. Subsequent detailed examination of the exome data suggested higher read depth at exons 45 and 46. Multiplex ligation-dependent probe amplification allowed identification of duplication of these two exons. This case suggests the potential usefulness of target exome sequencing in the prenatal diagnosis of the PKHD1 gene in ARPKD., Conclusions: This is the first report of intragenic duplication in the PKHD1 gene in ARPKD.
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- 2015
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16. Identification of novel FATP4 mutations in a Japanese patient with ichthyosis prematurity syndrome.
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Tsuge I, Morishita M, Kato T, Tsutsumi M, Inagaki H, Mori Y, Yamawaki K, Inuo C, Ieda K, Ohye T, Hayakawa A, and Kurahashi H
- Abstract
Ichthyosis prematurity syndrome (IPS) is a rare autosomal recessive disorder characterized by prematurity, a thick caseous scale at birth and lifelong atopic diathesis. Here, we describe the first Japanese case of IPS and report novel compound heterozygous mutations (p.C403Y and p.R510H) in fatty acid transport protein 4 (FATP4). She is the first reported patient of Asian origin, entirely distinct from the Scandinavian population, in whom the heterozygote carrier frequency is very high.
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- 2015
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17. Age-related decrease of meiotic cohesins in human oocytes.
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Tsutsumi M, Fujiwara R, Nishizawa H, Ito M, Kogo H, Inagaki H, Ohye T, Kato T, Fujii T, and Kurahashi H
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- Adult, Age Factors, Aneuploidy, Animals, Female, Humans, Mice, Middle Aged, Young Adult, Cohesins, Aging metabolism, Cell Cycle Proteins metabolism, Chromosomal Proteins, Non-Histone metabolism, Meiosis physiology, Oocytes metabolism
- Abstract
Aneuploidy in fetal chromosomes is one of the causes of pregnancy loss and of congenital birth defects. It is known that the frequency of oocyte aneuploidy increases with the human maternal age. Recent data have highlighted the contribution of cohesin complexes in the correct segregation of meiotic chromosomes. In mammalian oocytes, cohesion is established during the fetal stages and meiosis-specific cohesin subunits are not replenished after birth, raising the possibility that the long meiotic arrest of oocytes facilitates a deterioration of cohesion that leads to age-related increases in aneuploidy. We here examined the cohesin levels in dictyate oocytes from different age groups of humans and mice by immunofluorescence analyses of ovarian sections. The meiosis-specific cohesin subunits, REC8 and SMC1B, were found to be decreased in women aged 40 and over compared with those aged around 20 years (P<0.01). Age-related decreases in meiotic cohesins were also evident in mice. Interestingly, SMC1A, the mitotic counterpart of SMC1B, was substantially detectable in human oocytes, but little expressed in mice. Further, the amount of mitotic cohesins of mice slightly increased with age. These results suggest that, mitotic and meiotic cohesins may operate in a coordinated way to maintain cohesions over a sustained period in humans and that age-related decreases in meiotic cohesin subunits impair sister chromatid cohesion leading to increased segregation errors.
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- 2014
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18. Signature of backward replication slippage at the copy number variation junction.
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Ohye T, Inagaki H, Ozaki M, Ikeda T, and Kurahashi H
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- Abortion, Habitual genetics, Base Sequence, Chromosome Banding, Chromosome Deletion, Chromosomes, Human, Pair 2, Female, Humans, In Situ Hybridization, Fluorescence, Molecular Sequence Data, Chromosome Breakpoints, DNA Copy Number Variations, DNA Replication
- Abstract
Copy number abnormalities such as deletions and duplications give rise to a variety of medical problems and also manifest innocuous genomic variations. Aberrant DNA replication is suggested as the mechanism underlying de novo copy number abnormalities, but the precise details have remained unknown. In our present study, we analyzed the del(2)(q13q14.2) chromosomal junction site observed in a woman with a recurrent pregnancy loss. Microarray analyses allowed us to precisely demarcate a 2.8 Mb deletion in this case, which does not appear in the database of human genomic variations. This deletion includes only one brain-specific gene that could not be related to the reproduction failure of the patient. At the junction of the deletion, we found that 11-13-nucleotide sequence, originally located at the proximal breakpoint region, was repeated four times with a single-nucleotide microhomology at the joint between each repeat. The proximal region and the distal region was finally joined with six-nucleotide microhomology. The structure of the junction is consistent with backward replication slippage proposed previously. Our data lend support to the notion that a common DNA replication-mediated pathway generates copy number variation in the human genome.
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- 2014
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19. Dual roles for the telomeric repeats in chromosomally integrated human herpesvirus-6.
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Ohye T, Inagaki H, Ihira M, Higashimoto Y, Kato K, Oikawa J, Yagasaki H, Niizuma T, Takahashi Y, Kojima S, Yoshikawa T, and Kurahashi H
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- Base Sequence, DNA, Viral genetics, Genome, Viral genetics, Humans, Molecular Sequence Data, Chromosomes genetics, Herpesvirus 6, Human genetics, Repetitive Sequences, Nucleic Acid genetics, Telomere genetics, Virus Integration genetics
- Abstract
Approximately 1 percent of healthy individuals carry human herpesvirus-6 within a host chromosome. This is referred to as chromosomally integrated herpesvirus-6 (CIHHV-6). In this study, we investigated the chromosomal integration site in six individuals harboring CIHHV-6B. Using FISH, we found that HHV-6B signals are consistently located at the telomeric region. The proximal endpoints of the integrated virus were mapped at one of two telomere-repeat-like sequences (TRSs) within the DR-R in all cases. In two cases, we isolated junction fragments between the viral TRS and human telomere repeats. The distal endpoints were mapped at the distal TRS in all cases. The size of the distal TRS was found to be ~5 kb which is sufficient to fulfill cellular telomeric functions. We conclude that the viral TRS in the DR regions fulfill dual functions for CIHHV-6: homology-mediated integration into the telomeric region of the chromosome and neo-telomere formation that is then stably transmitted.
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- 2014
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20. A MEN2A family with two asymptomatic carriers affected by unilateral renal agenesis.
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Hibi Y, Ohye T, Ogawa K, Shimizu Y, Shibata M, Kagawa C, Mizuno Y, Kurahashi H, and Iwase K
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- Adrenal Gland Neoplasms diagnosis, Adrenal Gland Neoplasms genetics, Adrenal Gland Neoplasms surgery, Carcinoma, Medullary congenital, Carcinoma, Medullary genetics, Carcinoma, Medullary surgery, Congenital Abnormalities diagnosis, Female, Genotyping Techniques, Hirschsprung Disease genetics, Hirschsprung Disease surgery, Humans, Kidney Diseases diagnosis, Kidney Diseases genetics, Male, Middle Aged, Multiple Endocrine Neoplasia Type 2a surgery, Mutation, Pedigree, Pheochromocytoma diagnosis, Pheochromocytoma genetics, Pheochromocytoma surgery, Thyroid Neoplasms genetics, Thyroid Neoplasms surgery, Tomography, X-Ray Computed, Congenital Abnormalities genetics, Kidney abnormalities, Kidney Diseases congenital, Multiple Endocrine Neoplasia Type 2a genetics, Proto-Oncogene Proteins c-ret genetics
- Abstract
Accumulating evidences suggest RET gene's involvement in development of the kidney in mice and humans. Although it is well known that RET mutation causes multiple endocrine neoplasia type 2A (MEN2A), thus far only 3 individuals have been reported to have MEN2A and renal agenesis/dysgenesis. We report a MEN2A family with RET mutation in which two asymptomatic carriers presented with unilateral renal agenesis. A 48-year-old woman underwent total thyroidectomy with regional lymph node dissection in our department for medullary thyroid carcinoma. She had earlier surgical treatment for a left adrenal pheochromocytoma at the age of 45. In the screening for MEN type 2 for her three sons, a CT scan for adrenal pheochromocytoma incidentally found unilateral renal agenesis in two of the sons, one of whom had suffered from Hirschsprung's disease (HSCR). They had contralateral kidneys exhibiting compensatory hypertrophy and normal renal function. Genetic analysis detected C618R RET mutation in the proband and her 3 sons, and no other mutations were found in RET as well as glial cell line-derived neurotrophic factor (GDNF). Our data lend support to the hypothesis that constitutive active RET mutation in MEN type 2 might partially impair RET function and thereby cause loss of function phenotype such as renal agenesis or HSCR.
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- 2014
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21. Definition and refinement of the 7q36.3 duplication region associated with schizophrenia.
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Aleksic B, Kushima I, Ohye T, Ikeda M, Kunimoto S, Nakamura Y, Yoshimi A, Koide T, Iritani S, Kurahashi H, Iwata N, and Ozaki N
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- Adult, Aged, Case-Control Studies, Comparative Genomic Hybridization, DNA Copy Number Variations, Humans, In Situ Hybridization, Fluorescence, Middle Aged, Chromosome Duplication, Chromosomes, Human, Pair 7, Genetic Association Studies, Schizophrenia genetics
- Abstract
Using a very high-resolution oligonucleotide array for copy number variant (CNV) screening of samples comprising schizophrenic patients, we detected a novel CNV within the critical region (NCBI36/hg18, Chr7: 158,630,410-158,719,410) previously shown to be associated with schizophrenia. We investigated the association between the novel CNV identified in the current study and schizophrenia. Three independent samples were used: (1) Screening set, 300 Japanese schizophrenic patients (53.28 ± 14.66 years); (2) Confirmation set, 531 schizophrenic patients (46.03 ± 12.15 years); and (3) 711 healthy controls (47.12 ± 11.03 years). All subjects enrolled in the study were Japanese. Chromosomal position was determined using fluorescence in situ hybridization. We identified a novel duplication within the region associated with schizophrenia identified on 7q36.3 that is adjacent to VIPR2 and is not associated with schizophrenia. In the Japanese population, the 35-kb region that harbors the common, novel CNV should be excluded from the region associated with schizophrenia on 7q36.3.
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- 2013
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22. Two sequential cleavage reactions on cruciform DNA structures cause palindrome-mediated chromosomal translocations.
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Inagaki H, Ohye T, Kogo H, Tsutsumi M, Kato T, Tong M, Emanuel BS, and Kurahashi H
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- AT Rich Sequence, Base Sequence, Blotting, Southern, Chromosome Breakage, DNA, Cruciform chemistry, DNA, Cruciform genetics, DNA-Binding Proteins, Endonucleases, Gene Knockdown Techniques, Gene Rearrangement genetics, HEK293 Cells, Holliday Junction Resolvases metabolism, Humans, Male, Models, Biological, Molecular Sequence Data, Nuclear Proteins metabolism, Nucleotides genetics, Spermatozoa metabolism, DNA, Cruciform metabolism, Inverted Repeat Sequences genetics, Translocation, Genetic genetics
- Abstract
Gross chromosomal rearrangements (GCRs), such as translocations, deletions or inversions, are often generated by illegitimate repair between two DNA breakages at regions with nucleotide sequences that might potentially adopt a non-B DNA conformation. We previously established a plasmid-based model system that recapitulates palindrome-mediated recurrent chromosomal translocations in humans, and demonstrated that cruciform DNA conformation is required for the translocation-like rearrangements. Here we show that two sequential reactions that cleave the cruciform structures give rise to the translocation: GEN1-mediated resolution that cleaves diagonally at the four-way junction of the cruciform and Artemis-mediated opening of the subsequently formed hairpin ends. Indeed, translocation products in human sperm reveal the remnants of this two-step mechanism. These two intrinsic pathways that normally fulfil vital functions independently, Holliday-junction resolution in homologous recombination and coding joint formation in rearrangement of antigen-receptor genes, act upon the unusual DNA conformation in concert and lead to a subset of recurrent GCRs in humans.
- Published
- 2013
- Full Text
- View/download PDF
23. HORMAD2 is essential for synapsis surveillance during meiotic prophase via the recruitment of ATR activity.
- Author
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Kogo H, Tsutsumi M, Inagaki H, Ohye T, Kiyonari H, and Kurahashi H
- Subjects
- Animals, Apoptosis, Ataxia Telangiectasia Mutated Proteins, BRCA1 Protein metabolism, Cell Cycle Proteins genetics, DNA Breaks, Double-Stranded, Endodeoxyribonucleases deficiency, Endodeoxyribonucleases genetics, Female, Fertility genetics, Gene Silencing, Male, Meiotic Prophase I, Mice, Mice, Inbred C57BL, Mice, Inbred ICR, Mice, Knockout, Oocytes metabolism, Oocytes physiology, Ovary cytology, Ovary enzymology, Protein Transport, Spermatocytes metabolism, Spermatocytes physiology, Testis cytology, Cell Cycle Proteins metabolism, Cell Cycle Proteins physiology, Chromosome Pairing, Protein Serine-Threonine Kinases metabolism
- Abstract
Meiotic chromosome segregation requires homologous pairing, synapsis and crossover recombination during meiotic prophase. The checkpoint kinase ATR has been proposed to be involved in the quality surveillance of these processes, although the underlying mechanisms remain largely unknown. In our present study, we generated mice lacking HORMAD2, a protein that localizes to unsynapsed meiotic chromosomes. We show that this Hormad2 deficiency hampers the proper recruitment of ATR activity to unsynapsed chromosomes. Male Hormad2-deficient mice are infertile due to spermatocyte loss as a result of characteristic impairment of sex body formation; an ATR- and γH2AX-enriched repressive chromatin domain is formed, but is partially dissociated from the elongated sex chromosome axes. In contrast to males, Hormad2-deficient females are fertile. However, our analysis of Hormad2/Spo11 double-mutant females shows that the oocyte number is negatively correlated with the frequency of pseudo-sex body formation in a Hormad2 gene dosage-dependent manner. This result suggests that the elimination of Spo11-deficient asynaptic oocytes is associated with the HORMAD2-dependent pseudo-sex body formation that is likely initiated by local concentration of ATR activity in the absence of double-strand breaks. Our results thus show a HORMAD2-dependent quality control mechanism that recognizes unsynapsis and recruits ATR activity during mammalian meiosis., (© 2012 The Authors Genes to Cells © 2012 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.)
- Published
- 2012
- Full Text
- View/download PDF
24. Screening of genes involved in chromosome segregation during meiosis I: in vitro gene transfer to mouse fetal oocytes.
- Author
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Tsutsumi M, Kowa-Sugiyama H, Bolor H, Kogo H, Inagaki H, Ohye T, Yamada K, Taniguchi-Ikeda M, Toda T, and Kurahashi H
- Subjects
- Animals, Chromosome Pairing genetics, Embryonic Development, Female, Gene Expression Profiling, Male, Mice, Pregnancy, Recombination, Genetic, Spermatocytes cytology, Spermatocytes metabolism, Chromosome Segregation genetics, Gonads cytology, Gonads metabolism, Meiotic Prophase I genetics, Oocytes cytology, Oocytes metabolism
- Abstract
The events that take place during the prophase of meiosis I are essential for the correct segregation of homologous chromosomes. Defects in these processes likely contribute to infertility or recurrent pregnancy loss in humans. To screen for candidate genes for reproductive failure due to meiotic defects, we have analyzed the gene expression patterns in fetal, neonatal and adult gonads of both male and female mice by microarray and thereby identified 241 genes that are expressed specifically during prophase of meiosis I. Combined with our previous data obtained from developing spermatocytes, a total of 99 genes were identified that are upregulated in early prophase I. We confirmed the meiotic prophase I-specific expression of these genes using qRT-PCR. To further screen this panel for candidate genes that fulfill important roles in homologous pairing, synapsis and recombination, we established a gene transfer system for prophase I oocytes in combination with in vitro organ culture of ovaries, and successfully determined the localization of the selected genes. This gene set can thus serve as a resource for targeted sequence analysis via next-generation sequencing to identify the genes associated with human reproduction failure due to meiotic defects.
- Published
- 2012
- Full Text
- View/download PDF
25. Failure of homologous synapsis and sex-specific reproduction problems.
- Author
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Kurahashi H, Kogo H, Tsutsumi M, Inagaki H, and Ohye T
- Abstract
The prophase of meiosis I ensures the correct segregation of chromosomes to each daughter cell. This includes the pairing, synapsis, and recombination of homologous chromosomes. A subset of chromosomal abnormalities, including translocation and inversion, disturbs these processes, resulting in the failure to complete synapsis. This activates the meiotic pachytene checkpoint, and the gametes are fated to undergo cell cycle arrest and subsequent apoptosis. Spermatogenic cells appear to be more vulnerable to the pachytene checkpoint, and male carriers of chromosomal abnormalities are more susceptible to infertility. In contrast, oocytes tend to bypass the checkpoint and instead generate other problems, such as chromosome imbalance that often leads to recurrent pregnancy loss in female carriers. Recent advances in genetic manipulation technologies have increased our knowledge about the pachytene checkpoint and surveillance systems that detect chromosomal synapsis. This review focuses on the consequences of synapsis failure in humans and provides an overview of the mechanisms involved. We also discuss the sexual dimorphism of the involved pathways that leads to the differences in reproductive outcomes between males and females.
- Published
- 2012
- Full Text
- View/download PDF
26. HORMAD1-dependent checkpoint/surveillance mechanism eliminates asynaptic oocytes.
- Author
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Kogo H, Tsutsumi M, Ohye T, Inagaki H, Abe T, and Kurahashi H
- Subjects
- Animals, Cell Cycle Proteins genetics, Endodeoxyribonucleases genetics, Endodeoxyribonucleases metabolism, Female, Genes, cdc, Male, Mice, Mice, Knockout, Oocytes cytology, Oocytes metabolism, Phosphorylation, Spermatocytes cytology, Spermatocytes metabolism, Cell Cycle Proteins metabolism, Chromosome Pairing, Meiosis
- Abstract
Meiotic pachytene checkpoints monitor the failure of homologous recombination and synapsis to ensure faithful chromosome segregation during gamete formation. To date, the molecular basis of the mammalian pachytene checkpoints has remained largely unknown. We here report that mouse HORMAD1 is required for a meiotic prophase checkpoint that eliminates asynaptic oocytes. Hormad1-deficient mice are infertile and show an extensive failure of homologous pairing and synapsis, consistent with the evolutionarily conserved function of meiotic HORMA domain proteins. Unexpectedly, Hormad1-deficient ovaries contain a normal number of oocytes despite asynapsis and consequently produce aneuploid oocytes, indicating a checkpoint failure. By the analysis of Hormad1/Spo11 double mutants, the Hormad1 deficiency was found to abrogate the massive oocyte loss in the Spo11-deficient ovary. The Hormad1 deficiency also causes the eventual loss of pseudo sex body in the Spo11-deficient ovary and testis. These results suggest the involvement of HORMAD1 in the repressive chromatin domain formation that is proposed to be important in the meiotic prophase checkpoints. We also show the extensive phosphorylation of HORMAD1 in the Spo11-deficient testis and ovary, suggesting an involvement of novel DNA damage-independent phosphorylation signaling in the surveillance mechanism. Our present results provide clues to HORMAD1-dependent checkpoint in response to asynapsis in mammalian meiosis., (© 2012 The Authors. Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.)
- Published
- 2012
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- View/download PDF
27. Mechanism of complex gross chromosomal rearrangements: a commentary on concomitant microduplications of MECP2 and ATRX in male patients with severe mental retardation.
- Author
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Kurahashi H, Ohye T, Inagaki H, Kogo H, and Tsutsumi M
- Subjects
- Female, Humans, Male, DNA Helicases genetics, Gene Duplication genetics, Intellectual Disability genetics, Methyl-CpG-Binding Protein 2 genetics, Nuclear Proteins genetics
- Published
- 2012
- Full Text
- View/download PDF
28. DNA secondary structure is influenced by genetic variation and alters susceptibility to de novo translocation.
- Author
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Kato T, Inagaki H, Tong M, Kogo H, Ohye T, Yamada K, Tsutsumi M, Emanuel BS, and Kurahashi H
- Abstract
Background: Cumulative evidence suggests that DNA secondary structures impact DNA replication, transcription and genomic rearrangements. One of the best studied examples is the recurrent constitutional t(11;22) in humans that is mediated by potentially cruciform-forming sequences at the breakpoints, palindromic AT-rich repeats (PATRRs). We previously demonstrated that polymorphisms of PATRR sequences affect the frequency of de novo t(11;22)s in sperm samples from normal healthy males. These studies were designed to determine whether PATRR polymorphisms affect DNA secondary structure, thus leading to variation in translocation frequency., Methods: We studied the potential for DNA cruciform formation for several PATRR11 polymorphic alleles using mobility shift analysis in gel electrophoresis as well as by direct visualization of the DNA by atomic force microscopy. The structural data for various alleles were compared with the frequency of de novo t(11;22)s the allele produced., Results: The data indicate that the propensity for DNA cruciform structure of each polymorphic allele correlates with the frequency of de novo t(11;22)s produced (r = 0.77, P = 0.01)., Conclusions: Although indirect, our results strongly suggest that the PATRR adopts unstable cruciform structures during spermatogenesis that act as translocation hotspots in humans.
- Published
- 2011
- Full Text
- View/download PDF
29. Characterization of a novel mouse gene encoding an SYCP3-like protein that relocalizes from the XY body to the nucleolus during prophase of male meiosis I.
- Author
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Tsutsumi M, Kogo H, Kowa-Sugiyama H, Inagaki H, Ohye T, and Kurahashi H
- Subjects
- Amino Acid Sequence, Animals, Cell Cycle Proteins, Chromatin metabolism, Chromosomal Proteins, Non-Histone genetics, DNA-Binding Proteins, Female, Genes, X-Linked, Male, Mice, Molecular Sequence Data, Nuclear Proteins, RNA, Messenger metabolism, Sequence Homology, Amino Acid, Spermatogenesis, Up-Regulation, Cell Nucleolus metabolism, Chromosomal Proteins, Non-Histone metabolism, Meiotic Prophase I, Spermatocytes metabolism
- Abstract
Xlr6 is a novel but uncharacterized X-linked gene that is upregulated in meiotic prophase I during mouse spermatogenesis. Xlr6 belongs to the Xlr gene family, which includes a component of the axial/lateral element of the synaptonemal complex, Sycp3, and its transcripts are abundant in the fetal ovary and adult testis. Immunostaining and Western blot analysis demonstrate a diffuse localization pattern for this protein in the nucleus and an association with chromatin during the leptotene and zygotene stages. In males, XLR6 accumulates at the XY body of early pachytene to midpachytene spermatocytes, although the Xlr6 gene is subjected to meiotic sex chromosome inactivation. During the late pachytene and diplotene stages, the XLR6 protein relocalizes from the XY body to the nucleolus and, eventually, disappears by diakinesis. In females, XLR6 disappears at the pachytene stage, whereas it accumulates at the unpaired chromosomes occasionally observed in wild-type female mice. Although the amino acid sequence of XLR6 has a high similarity with SYCP3, its distinct localization pattern and dynamism suggest a unique chromatin modification function that leads to the transcriptional repression of ribosomal DNA in addition to sex chromosome genes.
- Published
- 2011
- Full Text
- View/download PDF
30. Paternal origin of the de novo constitutional t(11;22)(q23;q11).
- Author
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Ohye T, Inagaki H, Kogo H, Tsutsumi M, Kato T, Tong M, Macville MV, Medne L, Zackai EH, Emanuel BS, and Kurahashi H
- Subjects
- Alleles, Family, Female, Humans, Male, Pedigree, Polymerase Chain Reaction, Chromosomes, Human, Pair 11 genetics, Chromosomes, Human, Pair 22 genetics, Parents, Translocation, Genetic genetics
- Abstract
The constitutional t(11;22)(q23;q11) is a well-known recurrent non-Robertsonian translocation in humans. Although translocations generally occur in a random fashion, the break points of t(11;22)s are concentrated within several hundred base pairs on 11q23 and 22q11. These regions are characterized by palindromic AT-rich repeats (PATRRs), which appear to be responsible for the genomic instability. Translocation-specific PCR detects de novo t(11;22)s in sperm from healthy males at a frequency of 1/10(4)-10(5), but never in lymphoblasts, fibroblasts or other human somatic cell lines. This suggests that the generation of t(11;22) rearrangement is linked to gametogenesis, although female germ cells have not been tested. Here, we have studied eight cases of de novo t(11;22) to determine the parental origin of the translocation using the polymorphisms on the relevant PATRRs. All of the eight translocations were found to be of paternal origin. This result implicates a possible novel mechanism of sperm-specific generation of palindrome-mediated chromosomal translocations.
- Published
- 2010
- Full Text
- View/download PDF
31. Screening of genes involved in chromosome segregation during meiosis I: toward the identification of genes responsible for infertility in humans.
- Author
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Kogo H, Kowa-Sugiyama H, Yamada K, Bolor H, Tsutsumi M, Ohye T, Inagaki H, Taniguchi M, Toda T, and Kurahashi H
- Subjects
- Animals, Cell Line, Endodeoxyribonucleases, Esterases genetics, Esterases metabolism, Female, Fluorescent Antibody Technique, Gene Expression Profiling, Humans, In Situ Hybridization, Male, Meiotic Prophase I genetics, Mice, Mice, Inbred C57BL, Oligonucleotide Array Sequence Analysis, Reverse Transcriptase Polymerase Chain Reaction, Spermatocytes cytology, Spermatocytes metabolism, Testis cytology, Testis metabolism, Transfection, Chromosome Segregation genetics, Genetic Predisposition to Disease genetics, Infertility, Male genetics
- Abstract
Prophase I of male meiosis during early spermatogenesis involves dynamic chromosome segregation processes, including synapsis, meiotic recombination and cohesion. Genetic defects in the genes that participate in these processes consistently cause reproduction failure in mice. To identify candidate genes responsible for infertility in humans, we performed gene expression profiling of mouse spermatogenic cells undergoing meiotic prophase I. Cell fractions enriched in spermatogonia, leptotene/zygotene spermatocytes or pachytene spermatocytes from developing mouse testis were separately isolated by density gradient sedimentation and subjected to microarray analysis. A total of 726 genes were identified that were upregulated in leptotene/zygotene spermatocytes. To evaluate the screening efficiency for meiosis-specific genes, we randomly selected 12 genes from this gene set and characterized each gene product using reverse transcription (RT)-PCR of RNA from gonadal tissues, in situ hybridization on testicular tissue sections and subcellular localization analysis of the encoded protein. Four of the 12 genes were confirmed as genes expressed in meiotic stage and 2 of these 4 genes were novel, previously uncharacterized genes. Among the three confirmation methods that were used, RT-PCR appeared to be the most efficient method for further screening. These 726 candidates for human infertility genes might serve as a useful resource for next-generation sequencing combined with exon capture by microarray.
- Published
- 2010
- Full Text
- View/download PDF
32. Impaired DNA replication prompts deletions within palindromic sequences, but does not induce translocations in human cells.
- Author
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Kurahashi H, Inagaki H, Kato T, Hosoba E, Kogo H, Ohye T, Tsutsumi M, Bolor H, Tong M, and Emanuel BS
- Subjects
- Cell Line, DNA genetics, DNA metabolism, DNA Polymerase I genetics, DNA Polymerase I metabolism, Gene Expression Regulation, Humans, RNA, Small Interfering genetics, Translocation, Genetic, DNA Replication, Gene Deletion, Inverted Repeat Sequences
- Abstract
Palindromic regions are unstable and susceptible to deletion in prokaryotes and eukaryotes possibly due to stalled or slow replication. In the human genome, they also appear to become partially or completely deleted, while two palindromic AT-rich repeats (PATRR) contribute to known recurrent constitutional translocations. To explore the mechanism that causes the development of palindrome instabilities in humans, we compared the incidence of de novo translocations and deletions at PATRRs in human cells. Using a highly sensitive PCR assay that can detect single molecules, de novo deletions were detected neither in human somatic cells nor in sperm. However, deletions were detected at low frequency in cultured cell lines. Inhibition of DNA replication by administration of siRNA against the DNA polymerase alpha 1 (POLA1) gene or introduction of POLA inhibitors increased the frequency. This is in contrast to PATRR-mediated translocations that were never detected in similar conditions but were observed frequently in human sperm samples. Further deletions were found to take place during both leading- and lagging-strand synthesis. Our data suggest that stalled or slow replication induces deletions within PATRRs, but that other mechanisms might contribute to PATRR-mediated recurrent translocations in humans.
- Published
- 2009
- Full Text
- View/download PDF
33. Recent advance in our understanding of the molecular nature of chromosomal abnormalities.
- Author
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Kurahashi H, Bolor H, Kato T, Kogo H, Tsutsumi M, Inagaki H, and Ohye T
- Subjects
- Gene Duplication, Gene Rearrangement genetics, Genetic Predisposition to Disease, Humans, Meiosis, Translocation, Genetic, Chromosome Aberrations
- Abstract
The completion of the human genome project has enabled researchers to characterize the breakpoints for various chromosomal structural abnormalities including deletions, duplications or translocations. This in turn has shed new light on the molecular mechanisms underlying the onset of gross chromosomal rearrangements. On the other hand, advances in genetic manipulation technologies for various model organisms has increased our knowledge of meiotic chromosome segregation, errors which, contribute to chromosomal aneuploidy. This review focuses on the current understanding of germ line chromosomal abnormalities and provides an overview of the mechanisms involved. We refer to our own recent data and those of others to illustrate some of the new paradigms that have arisen in this field. We also discuss some perspectives on the sexual dimorphism of some of the pathways that leads to these chromosomal abnormalities.
- Published
- 2009
- Full Text
- View/download PDF
34. Chromosomal instability mediated by non-B DNA: cruciform conformation and not DNA sequence is responsible for recurrent translocation in humans.
- Author
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Inagaki H, Ohye T, Kogo H, Kato T, Bolor H, Taniguchi M, Shaikh TH, Emanuel BS, and Kurahashi H
- Subjects
- Cells, Cultured, Humans, Inverted Repeat Sequences genetics, Models, Biological, Molecular Sequence Data, Plasmids chemistry, Recurrence, Sequence Homology, Nucleic Acid, Base Sequence physiology, Chromosomal Instability genetics, DNA, Circular chemistry, DNA, Circular physiology, Nucleic Acid Conformation, Translocation, Genetic genetics
- Abstract
Chromosomal aberrations have been thought to be random events. However, recent findings introduce a new paradigm in which certain DNA segments have the potential to adopt unusual conformations that lead to genomic instability and nonrandom chromosomal rearrangement. One of the best-studied examples is the palindromic AT-rich repeat (PATRR), which induces recurrent constitutional translocations in humans. Here, we established a plasmid-based model that promotes frequent intermolecular rearrangements between two PATRRs in HEK293 cells. In this model system, the proportion of PATRR plasmid that extrudes a cruciform structure correlates to the levels of rearrangement. Our data suggest that PATRR-mediated translocations are attributable to unusual DNA conformations that confer a common pathway for chromosomal rearrangements in humans.
- Published
- 2009
- Full Text
- View/download PDF
35. Mutations of the SYCP3 gene in women with recurrent pregnancy loss.
- Author
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Bolor H, Mori T, Nishiyama S, Ito Y, Hosoba E, Inagaki H, Kogo H, Ohye T, Tsutsumi M, Kato T, Tong M, Nishizawa H, Pryor-Koishi K, Kitaoka E, Sawada T, Nishiyama Y, Udagawa Y, and Kurahashi H
- Subjects
- Adult, Cell Cycle Proteins, DNA-Binding Proteins, Female, Humans, Mutation, Pregnancy, Synaptonemal Complex genetics, Abortion, Habitual genetics, Genetic Predisposition to Disease, Nuclear Proteins genetics
- Abstract
Aneuploidy, a chromosomal numerical abnormality in the conceptus or fetus, occurs in at least 5% of all pregnancies and is the leading cause of early pregnancy loss in humans. Accumulating evidence now suggests that the correct segregation of chromosomes is affected by events occurring in prophase during meiosis I. These events include homologous chromosome pairing, sister-chromatid cohesion, and meiotic recombination. In our current study, we show that mutations in SYCP3, a gene encoding an essential component of the synaptonemal complex that is central to the interaction of homologous chromosomes, are associated with recurrent pregnancy loss. Two out of 26 women with recurrent pregnancy loss of unknown cause were found to carry independent heterozygous nucleotide alterations in this gene, neither of which was present among a group of 150 fertile women. Analysis of transcripts from minigenes harboring each of these two mutations revealed that both affected normal splicing, possibly resulting in the production of C-terminally mutated proteins. The mutant proteins were found to interact with their wild-type counterpart in vitro and inhibit the normal fiber formation of the SYCP3 protein when coexpressed in a heterologous system. These data suggest that these mutations are likely to generate an aberrant synaptonemal complex in a dominant-negative manner and contribute to abnormal chromosomal behavior that might lead to recurrent miscarriage. Combined with the fact that similar mutations have been previously identified in two males with azoospermia, our current data suggest that sexual dimorphism in response to meiotic disruption occurs even in humans.
- Published
- 2009
- Full Text
- View/download PDF
36. Age has no effect on de novo constitutional t(11;22) translocation frequency in sperm.
- Author
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Kato T, Yamada K, Inagaki H, Kogo H, Ohye T, Emanuel BS, and Kurahashi H
- Subjects
- Adult, Humans, Male, Middle Aged, Aging genetics, Spermatozoa physiology, Translocation, Genetic genetics
- Abstract
We analyzed de novo constitutional t(11;22) translocation frequency in sperm derived from normal healthy males as a function of the age of the sperm donors (from 25 to 51). Translocation-specific polymerase chain reaction demonstrated no age-dependent increment in the frequency of the rearrangements.
- Published
- 2007
- Full Text
- View/download PDF
37. Molecular cloning of a translocation breakpoint hotspot in 22q11.
- Author
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Kurahashi H, Inagaki H, Hosoba E, Kato T, Ohye T, Kogo H, and Emanuel BS
- Subjects
- AT Rich Sequence, Animals, Base Sequence, Chromosomes, Human, Pair 11, Chromosomes, Human, Pair 17, Cloning, Molecular, Cricetinae, DNA chemistry, DNA genetics, Humans, Hybrid Cells, Mice, Polymerase Chain Reaction, Repetitive Sequences, Nucleic Acid, Sequence Analysis, DNA, Chromosome Breakage, Chromosomes, Human, Pair 22 genetics, Translocation, Genetic
- Abstract
It has been well documented that 22q11 contains one of the most rearrangement-prone sites in the human genome, where the breakpoints of a number of constitutional translocations cluster. This breakage-sensitive region is located within one of the remaining unclonable gaps from the human genome project, suggestive of a specific sequence recalcitrant to cloning. In this study, we cloned a part of this gap and identified a novel 595-bp palindromic AT-rich repeat (PATRR). To date we have identified three translocation-associated PATRRs. They have common characteristics: (1) they are AT-rich nearly perfect palindromes, which are several hundred base pairs in length; (2) they possess non-AT-rich regions at both ends of the PATRR; (3) they display another nearby AT-rich region on one side of the PATRR. All of these features imply a potential for DNA secondary structure. Sequence analysis of unrelated individuals indicates no major size polymorphism, but shows minor nucleotide polymorphisms among individuals and cis-morphisms between the proximal and distal arms. Breakpoint analysis of various translocations indicates that double-strand-breakage (DSB) occurs at the center of the palindrome, often accompanied by a small symmetric deletion at the center. The breakpoints share only a small number of identical nucleotides between partner chromosomes. Taken together, these features imply that the DSBs are repaired through nonhomologous end joining or single-strand annealing rather than a homologous recombination pathway. All of these results support a previously proposed paradigm that unusual DNA secondary structure plays a role in the mechanism by which palindrome-mediated translocations occur.
- Published
- 2007
- Full Text
- View/download PDF
38. Genetic variation affects de novo translocation frequency.
- Author
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Kato T, Inagaki H, Yamada K, Kogo H, Ohye T, Kowa H, Nagaoka K, Taniguchi M, Emanuel BS, and Kurahashi H
- Subjects
- AT Rich Sequence, Alleles, Gene Frequency, Genotype, Heterozygote, Homozygote, Humans, Male, Repetitive Sequences, Nucleic Acid, Sequence Deletion, Genetic Variation, Spermatozoa, Translocation, Genetic
- Abstract
Translocation is one of the most frequently occurring human chromosomal aberrations. The constitutional t(11;22)(q23;q11), which is the only known recurrent non-Robertsonian translocation, represents a good model for studying translocations in humans. Here we demonstrate polymorphisms of the palindromic sequence at the t(11;22) breakpoint that affect the frequency of de novo translocations in sperm from normal males. A typical allele consists of a perfect palindrome, producing ~10-5 de novo t(11;22) translocations. Alleles with an asymmetric center do not form the t(11;22). Our data show the importance of genome sequence on chromosomal rearrangements, a class of human mutation that is thought to be random.
- Published
- 2006
- Full Text
- View/download PDF
39. Cruciform DNA structure underlies the etiology for palindrome-mediated human chromosomal translocations.
- Author
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Kurahashi H, Inagaki H, Yamada K, Ohye T, Taniguchi M, Emanuel BS, and Toda T
- Subjects
- Animals, Electrophoresis, Gel, Two-Dimensional, Genomic Instability, Humans, Nucleic Acid Conformation, Repetitive Sequences, Nucleic Acid genetics, DNA, Cruciform, Translocation, Genetic
- Abstract
There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoes a conformational change, causing temperature-dependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation.
- Published
- 2004
- Full Text
- View/download PDF
40. Catecholamines and serotonin are differently regulated by tetrahydrobiopterin. A study from 6-pyruvoyltetrahydropterin synthase knockout mice.
- Author
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Sumi-Ichinose C, Urano F, Kuroda R, Ohye T, Kojima M, Tazawa M, Shiraishi H, Hagino Y, Nagatsu T, Nomura T, and Ichinose H
- Subjects
- Animals, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphorus-Oxygen Lyases genetics, Biopterins analogs & derivatives, Biopterins physiology, Catecholamines genetics, Gene Expression Regulation physiology, Phosphorus-Oxygen Lyases physiology, Serotonin genetics
- Abstract
(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.
- Published
- 2001
- Full Text
- View/download PDF
41. A novel strategy for the negative selection in mouse embryonic stem cells operated with immunotoxin-mediated cell targeting.
- Author
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Kobayashi K, Ohye T, Pastan I, and Nagatsu T
- Subjects
- Animals, Feasibility Studies, Genetic Vectors, Humans, Mice, Mice, Transgenic, Mutagenesis, Site-Directed, Plasmids metabolism, Promoter Regions, Genetic, Pseudomonas, Receptors, Interleukin-2 genetics, Receptors, Interleukin-2 immunology, Receptors, Interleukin-2 metabolism, Pseudomonas aeruginosa Exotoxin A, ADP Ribose Transferases, Bacterial Toxins, Cell Separation methods, Exotoxins, Immunotoxins, Stem Cells cytology, Virulence Factors
- Abstract
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.
- Published
- 1996
- Full Text
- View/download PDF
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