-
[show abstract]
[hide abstract]
ABSTRACT: When the whole human genome sequence was determined by the Human Genome Project, the number of identified genes was fewer than expected. However, recent studies suggest that undiscovered transcripts still exist in the human genome. Furthermore, a new technology, the DNA microarray, which can simultaneously characterize huge amounts of genome sequence data, has become a useful tool for analyzing genetic changes in various diseases. A version of this tool, the tiling DNA microarray, was designed to search all the transcripts of the entire human genome, and provides huge amounts of data, including both exon and intron sequences, by a simple process. Although some previous studies using tiling DNA microarray analysis have indicated that numerous novel transcripts can be found in the human genome, none of them has reported any novel full-length human genes. Here, to find novel genes, we analyzed all the transcripts expressed in normal human prostate cells using this microarray. Because the optimal analytical parameters for using tiling DNA microarray data for this purpose had not been established, we established parameters for extracting the most likely regions for novel transcripts. The three parameters we optimized were the threshold for positive signal intensity, the Max gap, and the Min run, which we set to detect all transcriptional regions that were above the average length of known exons and had a signal intensity in the top 5%. We succeeded in obtaining the full-length sequence of one novel gene, located on chromosome 12q24.13. We named the novel gene "POTAGE". Its 5,841-bp mRNA consists of 26 exons. We detected part of exon 2 in the tiling data analysis. The full-length sequence was then obtained by RT-PCR and RACE. Although the function of POTAGE is unclear, its sequence showed high homology with genes in other species, suggesting it might have an important or essential function. This study demonstrates that the tiling DNA microarray can be useful for identifying novel human genes.
Gene 12/2012; · 2.34 Impact Factor
-
Masakazu Nakano,
Yoko Ikeda,
Yuichi Tokuda,
Masahiro Fuwa,
Natsue Omi,
Morio Ueno,
Kojiro Imai,
Hiroko Adachi,
Masaaki Kageyama,
Kazuhiko Mori,
Shigeru Kinoshita,
Kei Tashiro
[show abstract]
[hide abstract]
ABSTRACT: To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated.
We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8 × 10(-10)). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)--POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)--and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients.
In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.
PLoS ONE 01/2012; 7(3):e33389. · 4.09 Impact Factor
-
Journal of Human Genetics 08/2011; 56(8):550-1. · 2.57 Impact Factor
-
Mayumi Ueta,
Chie Sotozono, Masakazu Nakano,
Takazumi Taniguchi,
Tomohito Yagi,
Yuichi Tokuda,
Masahiro Fuwa,
Tsutomu Inatomi,
Norihiko Yokoi,
Kei Tashiro,
Shigeru Kinoshita
[show abstract]
[hide abstract]
ABSTRACT: Stevens-Johnson syndrome (SJS) and its severe variant, toxic epidermal necrolysis (TEN), are acute inflammatory vesiculobullous reactions of the skin and mucosa. They often affect the ocular surface and can result in permanent visual dysfunction.
We sought to discover genetic markers for SJS/TEN susceptibility.
We performed a genome-wide association study with 60 patients and 300 control subjects. We applied stringent filter and visual assessments for selecting single nucleotide polymorphisms (SNPs) and a high false discovery rate threshold. We fine-mapped the region where a candidate SNP was found and confirmed the results by means of sequencing. We evaluated the function of agonist-activated prostaglandin E receptor 3 (EP3), the gene for which contained several SNPs, in regulating cytokine production in human conjunctival epithelial (CE) cells. The expression levels of EP3 in the CE cells from patients and control subjects were also compared.
We identified 3 SNPs that passed the false discovery rate threshold. One (rs17131450) was close to the EP3 gene. Therefore we analyzed the EP3 region in detail and identified 5 other SNPs. We confirmed the association between SJS/TEN and all 6 SNPs. Activated EP3 was expressed in control CE cells, and it suppressed polyI:C-stimulated cytokine production, suggesting that EP3 might help prevent ocular surface inflammation. Concordantly, the EP3 levels were much lower in the CE cells of the patients than in those of the control subjects.
We demonstrated, using both genetic and functional analyses, that EP3 could be a key player in the pathogenesis of SJS/TEN accompanied by ocular complications.
The Journal of allergy and clinical immunology 10/2010; 126(6):1218-25.e10. · 9.17 Impact Factor
-
Masakazu Nakano,
Yoko Ikeda,
Takazumi Taniguchi,
Tomohito Yagi,
Masahiro Fuwa,
Natsue Omi,
Yuichi Tokuda,
Masami Tanaka,
Kengo Yoshii,
Masaaki Kageyama,
Shigeta Naruse,
Akira Matsuda,
Kazuhiko Mori,
Shigeru Kinoshita,
Kei Tashiro
[show abstract]
[hide abstract]
ABSTRACT: Primary open-angle glaucoma (POAG) is the major type of glaucoma. To discover genetic markers associated with POAG, we examined a total of 1,575 Japanese subjects in a genome-wide association study (stage 1) and a subsequent study (stage 2). Both studies were carried out at a single institution. In the stage 1 association study, we compared SNPs between 418 POAG patients and 300 control subjects. First, low-quality data were eliminated by a stringent filter, and 331,838 autosomal SNPs were selected for analysis. Poorly clustered SNPs were eliminated by a visual assessment, leaving 255 that showed a significant deviation (P < 0.001) in the allele frequency comparison. In the stage 2 analysis, we tested these 255 SNPs for association in DNA samples from a separate group of 409 POAG and 448 control subjects. High-quality genotype data were selected and used to calculate the combined P values of stages 1 and 2 by the Mantel-Haenszel test. These analyses yielded 6 SNPs with P < 0.0001. All 6 SNPs showed a significant association (P < 0.05) in stage 2, demonstrating a confirmed association with POAG. Although we could not link the SNPs to the annotated gene(s), it turned out that we have identified 3 genetic loci probably associated with POAG. These findings would provide the foundation for future studies to build on, such as for the metaanalysis, to reveal the molecular mechanism of the POAG pathogenesis.
Proceedings of the National Academy of Sciences 08/2009; 106(31):12838-42. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Stromal cell-derived factor 1 (SDF-1; CXCL12/pre-B cell growth-stimulating factor) is a dominant chemokine in bone marrow and is known to be involved in inflammatory diseases, including rheumatoid arthritis. However, its role in bone repair remains unknown. The purpose of this study was to investigate the role of SDF-1 and its receptor, CXCR4, in bone healing.
The expression of SDF-1 during the repair of a murine structural femoral bone graft was examined by real-time polymerase chain reaction and immunohistochemical analysis. The bone graft model was treated with anti-SDF-1 neutralizing antibody or TF14016, an antagonist for CXCR4, and evaluated by histomorphometry. The functional effect of SDF-1 on primary mesenchymal stem cells was determined by in vitro and in vivo migration assays. New bone formation in an exchanging-graft model was compared with that in the autograft models, using mice partially lacking SDF-1 (SDF-1(+/-)) or CXCR4 (CXCR4(+/-)).
The expression of SDF1 messenger RNA was increased during the healing of live bone grafts but was not increased in dead grafts. High expression of SDF-1 protein was observed in the periosteum of the live graft. New bone formation was inhibited by the administration of anti-SDF-1 antibody or TF14016. SDF-1 increased mesenchymal stem cell chemotaxis in vitro in a dose-dependent manner. The in vivo migration study demonstrated that mesenchymal stem cells recruited by SDF-1 participate in endochondral bone repair. Bone formation was decreased in SDF-1(+/-) and CXCR4(+/-) mice and was restored by the graft bones from CXCR4(+/-) mice transplanted into the SDF-1(+/-) femur, but not vice versa.
SDF-1 is induced in the periosteum of injured bone and promotes endochondral bone repair by recruiting mesenchymal stem cells to the site of injury.
Arthritis & Rheumatism 03/2009; 60(3):813-23. · 7.87 Impact Factor
-
Kazuhiko Mori,
Kojiro Imai,
Akira Matsuda,
Yoko Ikeda,
Shigeta Naruse,
Hisako Hitora-Takeshita, Masakazu Nakano,
Takazumi Taniguchi,
Natsue Omi,
Kei Tashiro,
Shigeru Kinoshita
[show abstract]
[hide abstract]
ABSTRACT: We performed genetic association studies using a native Japanese population to examine the reproducibility of results of lysyl oxidase-like 1 (LOXL1) genetic association studies for exfoliation glaucoma (XFG) beyond the differences of ethnicity. We also quantified LOXL1 mRNA expression in the human lens capsule to examine the possible correlation between LOXL1 expression and XFG pathogenesis.
We performed a case-control study using 95 Japanese XFG patients and 190 controls. Real-time polymerase chain reaction (PCR) analysis was performed using lens capsules obtained during surgery.
The TT genotype in the single nucleotide polymorphism (SNP) rs1048661 and the GG genotype in the SNP rs3825942 in exon 1 of LOXL1 were significantly associated with an increased risk of XFG under recessive models (chi(2) test, p=5.34 x 10(-34) and p=2.1 x 10(-8), respectively). Quantification of LOXL1 mRNA expression demonstrated no significant difference between XFG and senile cataract samples.
Although the functional effects of the LOXL1 SNP appear to be qualitative rather than quantitative, the amino acid substitution (R141L) caused by SNP rs1048661 is not a simple decisive factor for XFG due to the inverted allele frequency between Japanese XFG and Caucasian XFG patients. Further genetic and functional studies are essential for clarifying XFG pathogenesis.
Molecular vision 02/2008; 14:1037-40. · 2.20 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: TGF-beta3 has been implicated in the pathology of ocular diseases, but its concentration in human aqueous humor has never been assessed. In this study, we established an enzyme-linked immunosorbent assay (ELISA) for TGF-beta3 and quantitated it in aqueous humor collected from patients with pseudoexfoliation syndrome (PE), primary open angle glaucoma, chronic angle closure glaucoma and cataract (as the control). To develop the TGF-beta3 ELISA, we screened antibodies to identify the best combination, validated the assay for aqueous humor, and optimized the procedure for preparing activated TGF-beta3. As a result, our ELISA was highly selective and reproducible. Using our ELISA, we discovered a significantly elevated concentration of TGF-beta3 in PE eyes. We also developed new TGF-beta1 and -beta2 ELISAs, and were able, for the first time, to quantitate all the TGF-beta isoforms in the aqueous humor from a single eye, to assess their proportional effects on the pathogenesis of ocular diseases.
Growth Factors 07/2007; 25(3):160-7. · 1.65 Impact Factor
-
