Hiroshi Inoue

Kanagawa Children's Medical Center, Yokohama, Kanagawa, Japan

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Publications (37)128.49 Total impact

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    ABSTRACT: TMEM16E/GDD1 has been shown to be responsible for the bone-related late-onset disease gnathodiaphyseal dysplasia (GDD), with the dominant allele (TMEM16E(gdd) ) encoding a missense mutation at Cys356. Additionally, several recessive loss-of-function alleles of TMEM16E also cause late-onset limb girdle muscular dystrophy. In this study, we found that TMEM16E was rapidly degraded via the proteasome pathway, which was rescued by inhibition of the PI3K pathway and by the chemical chaperone, sodium butyrate. Moreover, TMEM16E(gdd) exhibited lower stability than TMEM16E, but showed similar propensity to be rescued. TMEM16E did not exhibit cell surface calcium-dependent chloride channel (CaCC) activity, which was originally identified in TMEM16A and TMEM16B, due to their intracellular vesicle distribution. A putative pore-forming domain of TMEM16E, which shared 39.8% similarity in 98 amino acids with TMEM16A, disrupted CaCC activity of TMEM16A via domain swapping. However, the Thr611Cys mutation in the swapped domain, which mimicked conserved cysteine residues between TMEM16A and TMEM16B, reconstituted CaCC activity. In addition, the GDD-causing cysteine mutation made in TMEM16A drastically altered CaCC activity. Based on these findings, TMEM16E possesses distinct function other than CaCC and another protein-stabilizing machinery toward the TMEM16E and TMEM16E(gdd) proteins should be considered for the on-set regulation of their phenotypes in tissues. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
    Journal of Cellular Physiology 07/2013; · 4.22 Impact Factor
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    ABSTRACT: OBJECTIVE: To elucidate the pathogenesis of isolated growth hormone (GH) deficiency in a Japanese girl without consanguinity. DESIGN: A 2-year-old girl of height 77.2cm (-3.0 SD for Japanese girls) was found to have an insulin-like growth factor (IGF)-1 level of 7ng/mL and IGF binding protein-3 (IGFBP-3) level of 0.41μg/mL. GH responded modestly to a series of pharmacological stimulants, increasing to 2.81ng/mL with insulin-induced hypoglycemia, 3.78ng/mL with arginine, and 3.93 with GH-releasing hormone (GHRH). Following direct sequencing of the GHRH receptor (GHRHR) gene, evaluation by the luciferase reporter assay, immunofluorescence study, and in vitro splicing assay with minigene constructs was conducted. RESULTS: Novel compound heterozygous GHRHR gene mutations were identified in the patient. A p.G136V substitution elicited no luciferase activity increment in response to GHRH stimulation, with normal membranous expression. Splicing assay demonstrated that the IVS2+3a>g mutation would lead to aberrant splicing. CONCLUSIONS: A case of isolated GH deficiency due to novel GHRHR gene mutations was identified.
    Growth hormone & IGF research: official journal of the Growth Hormone Research Society and the International IGF Research Society 04/2013; · 2.35 Impact Factor
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    ABSTRACT: We have previously identified significant quantitative trait loci (QTL) Dbm1 (diabetic modifier QTL 1) on chromosome 6, affecting plasma glucose and insulin concentrations and body weight on F(2) progeny of hypoinsulinemic diabetic Akita mice, with the heterozygous Ins2 gene Cys96Tyr mutation, and non-diabetic A/J mice. To discover diabetic modifier genes on Dbm1, we constructed congenic strain for Dbm1 using the Akita allele as donor in A/J allele genetic background, and compared diabetes-related phenotypes to control mice. The homozygote for Akita allele of Dbm1 was associated with lower plasma glucose concentrations in glucose tolerance test (GTT) in the hypoinsulinemic condition derived from the Ins2 mutation and lower plasma insulin concentrations and body weight in the normoinsulinemic condition without the Ins2 mutation than the homozygote for A/J allele, as we performed QTL analysis on F(2) intercross mice. The Akita allele also decreased the epididymal white adipose tissue (EWAT) weight. According to the analysis of sub-congenic strains, we narrowed down the responsible diabetic modifier region to 9 Mb. As fourteen candidate genes exist in this region, we analyzed genomic variants of these genes and gene expression in the muscle, liver, and EWAT and identified that Bhlhe40 gene expression in muscle is decreased in congenic mice. According to the in vitro functional analyses, Bhlhe40 was shown to negatively control fatty acid oxidation in cultured myocyte. Based on these, we conclude that Bhlhe40 is a possible candidate diabetic modifier gene responsible for Dbm1 locus affecting diabetes and/or obesity through negatively controlling fatty acid oxidation in muscle.
    Genes & Genetic Systems 01/2012; 87(4):253-64. · 1.13 Impact Factor
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    ABSTRACT: We have previously identified four significant quantitative trait loci (QTL) affecting plasma glucose concentrations on F(2) progeny of hypoinsulinemic diabetic Akita mice, heterozygous for the Ins2 gene Cys96Tyr mutation, and non-diabetic A/J mice, one of which on chromosome 15 named Dbm4 (diabetic modifier QTL 4) was shown to affect fasting plasma glucose concentrations with a maximum LOD score of 6.17. To estimate the influence of Dbm4 itself to the diabetes-related phenotypes, we constructed congenic strain with heterozygous Ins2 mutation using the Akita allele as donor of Dbm4 locus in the A/J genetic background, and measured quantitative traits including plasma glucose concentrations in glucose tolerance test (GTT). In this study, we found the Akita allele in Dbm4 was associated with higher fasting plasma glucose concentrations as in previous QTL analysis. According to gene expression assay, key enzymes of hepatic gluconeogenesis were expressed to the more increased degree in the liver of congenic mice compared to the A/J allele based control mice. Based on these results, we concluded that diabetic modifier gene(s) exist on Dbm4 locus affecting fasting plasma glucose concentrations via regulation of gluconeogenic gene expression in the hypoinsulinemic diabetic condition. Identification of the modifier gene responsible for Dbm4 would provide new drug development targets for human type 2 diabetes with hepatic insulin resistance.
    Genes & Genetic Systems 01/2012; 87(5):341-6. · 1.13 Impact Factor
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    ABSTRACT: To date, approximately 35 different POU1F1 mutations have been described in patients with familial and sporadic combined pituitary hormone deficiency (CPHD) from different ethnic backgrounds. The majority are missense mutations clustered within the conserved POU-specific and POU-homeo domains, encoded by exons 4 and 6, respectively. This study aimed to identify the molecular basis and clinical characteristics of a Japanese CPHD family with a novel POU1F1 mutation. The POU1F1 gene was sequenced in identical twin brothers with mild CPHD. The mutation identified was also evaluated in family members as well as 188 Japanese controls and then examined in functional studies. A novel heterozygous splice site mutation (Ex2 + 1G>T; c.214 + 1G>T) was detected. This mutation was also present in their undiagnosed mother, but not in any of the controls. In vitro splicing studies suggested this mutation to result in an in-frame skipping of exon 2, thus producing an internally deleted protein lacking most of the R2 transactivation subdomain (TAD-R2). Heterologous expression studies of the mutated POU1F1 protein showed only modest reductions in its transactivation activities in HEK293T cells, while acting as a dominant-negative inhibitor of the endogenous activities of POU1F1 in pituitary GH3 cells. This is the first report of a mutation at the exon 2 donor splice site of POU1F1, affecting TAD-R2. The addition of this mutation to the growing list of pathological POU1F1 mutations may provide deeper insights into clinical heterogeneity in the expressions of individual mutations and a better understanding of the structure-function relationships of POU1F1.
    Clinical Endocrinology 07/2011; 76(1):78-87. · 3.40 Impact Factor
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    ABSTRACT: In the current study, to elucidate the molecular basis of cell type-specific expression of the GH-secretagogue/ghrelin receptor type 1A (GHSR1A), we characterized the structure and putative promoter region of the rat Ghsr gene. We identified an alternative 5'-untranslated first exon that contains multiple transcription start sites, and confirmed a 200-bp sequence proximal to this exon to be sufficient for basal promoter activity. A promoter-associated CpG island conserved across different species was found to be hypomethylated in Ghsr1a-expressing cell lines, while being heavily methylated in non-expressing cells. In cells with low or absent Ghsr1a expression, treatment with demethylating agents activated Ghsr1a transcription. Chromatin immunoprecipitation assays demonstrated Ghsr1a-expressing cells to display active histone modifications, whereas repressive modifications were present exclusively in other cell types. These results suggest epigenetic modifications at GHSR to play important roles in determining GHSR1A expression and abundance, and therefore the consequent sensitivity of cells to ghrelin.
    Molecular and Cellular Endocrinology 07/2011; 345(1-2):1-15. · 4.04 Impact Factor
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    ABSTRACT: Short stature (SS) is a multifactorial developmental condition with a significant genetic component. Recent studies have revealed that rare deleterious mutations in the GH-secretagogue receptor type 1A (GHSR1A) gene could be a cause of familial SS or GH deficiency. The aim of this study was to evaluate the contribution of GHSR1A mutations to the molecular mechanism underlying SS in Japanese subjects. We performed mutational screening of the GHSR1A gene in 127 unrelated Japanese SS patients diagnosed with either isolated GH deficiency or idiopathic SS. Identified mutations were analyzed in 188 control subjects, and their functional properties were examined in a heterologous expression system. Four novel heterozygous GHSR1A mutations were identified (ΔQ36, P108L, C173R, and D246A). Expression studies demonstrated that these mutations had varying functional consequences: 1) all mutations showed a loss-of-function effect on the constitutive signaling activity of GHSR1A, but the degree of loss varied widely; 2) C173R caused intracellular retention of the mutated protein, resulting in total loss of receptor function; 3) P108L resulted in a large decrease in binding affinity to ghrelin, without affecting its surface expression; 4) D246A uniquely impaired agonist- and inverse agonist-stimulated receptor signaling; and 5) ΔQ36 showed only a subtle reduction in constitutive activity. The cumulative frequency of these putative functional mutations was significantly higher in the patient group than in controls (4.72 vs. 0.53%; P = 0.019; odds ratio = 9.28; 95% confidence interval, 1.10-78.0). Our results suggest that GHSR1A mutations contribute to the genetic etiology of SS in the Japanese population.
    The Journal of Clinical Endocrinology and Metabolism 11/2010; 96(2):E373-8. · 6.31 Impact Factor
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    ABSTRACT: Growth hormone-releasing hormone receptor (GHRHR) gene mutations have been identified in patients of different ethnic origins with isolated GH deficiency (IGHD) type IB. However, the prevalence of these mutations in the Japanese population has yet to be fully determined. This study aimed to evaluate the contributions of GHRHR mutations to the molecular mechanism underlying short stature in Japanese subjects. The GHRHR gene was sequenced in 127 unrelated Japanese patients with either IGHD (n = 14) or idiopathic short stature (ISS; n = 113). Sequence variants were evaluated in family members and 188 controls, and then examined in functional studies. A novel homozygous E382E (c.1146G>A) synonymous variant, at the last base of exon 12, was identified in an IGHD family with two affected sisters. In vitro splicing studies showed this mutation to result in skipping of exon 12. In one ISS patient, a heterozygous ATG-166T>C variant was found in the distal Pit-1 P2 binding element of the GHRHR promoter. In two control subjects, a close but distinct variant, ATG-164T>C, was detected. Functional studies showed that both promoter variants diminish promoter activity by altering Pit-1 binding ability. Four missense variants were also found in both patient and control groups but had no detectable functional consequences. The homozygous GHRHR mutation was rare, being detected in only one Japanese IGHD family. Future research is needed to clarify the genetic contributions of heterozygous functional promoter variants to GHD, ISS and normal-stature variations.
    Clinical Endocrinology 11/2010; 74(2):223-33. · 3.40 Impact Factor
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    ABSTRACT: The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.
    The American Journal of Human Genetics 02/2010; 86(2):213-21. · 11.20 Impact Factor
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    ABSTRACT: High-mobility group A2 is highly expressed during embryogenesis and in various benign and malignant tumors. Recent studies report that high-mobility group A2 is negatively regulated by the let-7 microRNAs (miRNAs) family in vitro. The development of pituitary adenomas in high-mobility group A2 transgenic mice showed that high-mobility group A2 may be involved in pituitary tumorigenesis. However, no studies have investigated the clinical significance of high-mobility group A2 and its relationship to the let-7 miRNA family in human pituitary adenomas. Using immunohistochemistry, we analyzed high-mobility group A2 expression with respect to various clinicopathologic factors in 98 pituitary adenomas. Overexpression of high-mobility group A2 was observed in 39% (38/98) of pituitary adenomas compared with normal adenohypophysial tissue and was frequently found in adenomas including prolactin (PRL), adrenocorticotrophic hormone, or follicle-stimulating hormone/luteinizing hormone and in null cell adenomas, but relatively rare in growth hormone (GH) and mixed GH/PRL adenomas. High-mobility group A2 expression was significantly associated with tumor invasion (P<0.05) and was significantly higher in grade IV than in grades I, II, and III adenomas (P<0.05). High levels of high-mobility group A2 expression were more frequently observed in macroadenomas than in microadenomas (P<0.05). High levels of high-mobility group A2 expression also significantly correlated with the proliferation marker Ki-67 (P<0.0001). Real-time quantitative RT-PCR analysis was carried out to evaluate the expression of let-7 in 55 pituitary adenomas. Subsequently, decreased expression of let-7 was confirmed in 23 of 55 (42%) adenomas and was correlated with high-grade tumors (P<0.05). An inverse correlation between let-7 and high-mobility group A2 expression was evident (R=−0.33, P<0.05). These findings support a causal link between let-7 and high-mobility group A2 whereby loss of let-7 expression induces high-mobility group A2 upregulation that represents an important mechanism in pituitary tumorigenesis and progression.Keywords: pituitary adenoma, HMGA2, let-7, tumorigenesis
    Modern Pathology 01/2009; 22(3):431-441. · 5.25 Impact Factor
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    ABSTRACT: Genetic variants of the transcription factor 7-like 2 (TCF7L2) gene affect the risk of type 2 diabetes in populations with multiple ethnic groups. However, a comprehensive survey of this gene has not been done for a Japanese population. Thus, we conducted this gene-based association study, in which the common genetic variants were analyzed. Using 24 Japanese type 2 diabetic subjects, we first screened a 9.5 kb region, which included the entire coding sequence, to assess potential functional variants of TCF7L2. Sequencing revealed a common coding variant (Pro477Thr) in exon 14 of TCF7L2 that was not enrolled in the public SNP database. Nineteen SNPs and the microsatellite DG10S478 were genotyped across the gene in 2,877 unrelated Japanese subjects. This independent screen identified the previously reported rs7903146 with a strongest association (allele P = 0.0001, odds ratio = 1.59 [95% confidence interval 1.25-2.01]), but there was no significant association between Pro477Thr and type 2 diabetes (allele P = 0.64). Expression of the Pro477Thr variant did not alter TCF7L2 expression in 30 lymphoblast cells. Although a genotypic effect of Pro477Thr on expression of TCF7L2 was not apparent, Pro477Thr was identified as a common variant of TCF7L2 in 2,877 Japanese subjects. Further functional studies are required to determine the possible effect of this coding variant on type 2 diabetes.
    Journal of Human Genetics 12/2008; 53(11-12):972-82. · 2.37 Impact Factor
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    ABSTRACT: In an attempt to rectify the hyperglycemic state in obese insulin resistant db/db mice, a transgenic line was generated (db/db-CDK4(R24C)) that expresses a constitutively active form of cyclin-dependent kinase 4 (CDK4/R24C) under the control of the insulin promoter. Compared with non-transgenic db/db littermates, adult db/db-CDK4(R24C) mice show near-complete glycemic normalization and improved plasma lipid concentrations, but are also more susceptible to weight gain and have significantly lower plasma adiponection levels. They have striking islet hypertrophy and beta-cell hyperplasia, and retain an insulin secretory response during the glucose tolerance test. We examined the expression of several key regulatory transcription factor genes involved in lipid and glucose metabolism in insulin target tissues of db/db-CDK4(R24C) as well as db/db mice, and found that the expression levels of members of the peroxisome proliferator-activated receptor (PPAR) family are highly associated with metabolic alterations in a gene- and tissue-specific manner. We show for the first time that the Ppar-delta in skeletal muscle and white adipose tissues is transcriptionally down-regulated in db/db mice. The db/db-CDK4(R24C) mice present a novel model of leptin-resistant obesity with compensatory hyperinsulinemia and normalized blood glucose levels, and thus may be useful for future studies that aim to dissect relationships between insulin and leptin signaling.
    Diabetes research and clinical practice 09/2008; 82(1):33-41. · 2.74 Impact Factor
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    ABSTRACT: STAT4 encodes a transcriptional factor that transmits signals induced by several key cytokines, and it might be a key molecule in the development of autoimmune diseases. Recently, a STAT4 haplotype was reported to be associated with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) in Caucasian populations. This was replicated in a Korean RA population. Interestingly, the degree of risk of RA susceptibility with the STAT4 haplotype was similar in the Caucasian and Korean populations. The present study was undertaken to investigate the effect of STAT4 on susceptibility to RA and SLE in the Japanese. We performed an association study using 3 independent Japanese RA case-control populations (total 3,567 cases and 2,199 controls) and 3 independent Japanese SLE populations (total 591 cases). All samples were genotyped using the TaqMan fluorogenic 5' nuclease assay for single-nucleotide polymorphism (SNP) rs7574865, which tags the susceptibility haplotype. The association of the SNP with disease susceptibility in each case-control study was calculated using Fisher's exact test, and the results were combined, using the Mantel-Haenszel method, to obtain combined odds ratios (ORs). We observed a significant association of the STAT4 polymorphism with susceptibility to both RA and SLE. The combined ORs for RA and SLE, respectively, were 1.27 (P = 8.4 x 10(-9)) and 1.61 (P = 2.1 x 10(-11)) for allele frequency distribution; these ORs were quite similar to those previously observed in the Caucasian population. We conclude that STAT4 is associated with RA and SLE in the Japanese. Our results indicate that STAT4 is a common genetic risk factor for autoimmune diseases, with similar strength across major racial groups.
    Arthritis & Rheumatology 08/2008; 58(7):1940-6. · 7.48 Impact Factor
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    ABSTRACT: The LKB1-AMPK-TORC2 signaling pathway controls glucose homeostasis in the liver, and mediates therapeutic effects of insulin-sensitizing antidiabetic agents. To examine whether genetic variations in genes encoding components of this signaling pathway contribute to increased susceptibility to type 2 diabetes, we screened STK11 (LKB1) and CRTC2 (TORC2) genes for genetic variants and conducted a case-control study in 1787 unrelated Japanese individuals. Additionally, the previously described association between the PRKAA2 (AMPK alpha2-subunit) haplotype and type 2 diabetes was tested for replication. We observed associations of nominal significance with two SNPs, an intronic SNP in the STK11 (rs741765; OR 1.33, 95% CI 1.05-1.67, p=0.017, under a recessive genetic model), and a non-synonymous SNP in the CRTC2 (6909C>T: Arg379Cys; OR 3.01, 95% CI 1.18-7.66, p=0.016, under a dominant model), although neither withstood correction for multiple testing. We were unable to replicate the association between the PRKAA2 haplotype and type 2 diabetes: however, in the single SNP evaluation, an intronic PRKAA2 SNP (rs1418442) that had previously been reported to be associated with serum cholesterol levels in Caucasian females showed a weak association (OR 0.62, 95% CI 0.40-0.96, p=0.030, under a recessive model). Among the three genes investigated herein, gene-gene (SNP-SNP) interaction studies provided evidence for an interaction between STK11 and CRTC2 influencing susceptibility to type 2 diabetes. Our findings suggest that genetic variants of LKB1-AMPK-TORC2 pathway components may exert a weak influence on the occurrence of type 2 diabetes in Japanese.
    Molecular Genetics and Metabolism 02/2008; 93(2):200-9. · 2.83 Impact Factor
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    ABSTRACT: Chromosome 15q14-22.1 has been linked to type 2 diabetes (T2D) and its related traits in Japanese and other populations. The presence of T2D disease susceptibility variant(s) was assessed in the 21.8 Mb region between D15S118 and D15S117 in a Japanese population using a region-wide case-control association test. A two-stage association test was performed using Japanese subjects: The discovery panel (Stage 1) used 372 cases and 360 controls, while an independent replication panel (Stage 2) used 532 cases and 530 controls. A total of 1,317 evenly-spaced, common SNP markers with minor allele frequencies > 0.10 were typed for each stage. Captured genetic variation was examined in HapMap JPT SNPs, and a haplotype-based association test was performed. SNP2140 (rs2412747) (C/T) in intron 33 of the ubiquitin protein ligase E3 component n-recognin 1 (UBR1) gene was selected as a landmark SNP based on repeated significant associations in Stage 1 and Stage 2. However, the marginal p value (p = 0.0043 in the allelic test, OR = 1.26, 95% CI = 1.07-1.48 for combined samples) was weak in a single locus or haplotype-based association test. We failed to find any significant SNPs after correcting for multiple testing. The two-stage association test did not reveal a strong association between T2D and any common variants on chromosome 15q14-22.1 in 1,794 Japanese subjects. A further association test with a larger sample size and denser SNP markers is required to confirm these observations.
    BMC Medical Genetics 01/2008; 9:22. · 2.54 Impact Factor
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    ABSTRACT: We conducted population-based association tests for the four selected SNPs (rs2240340/padi4_94, rs7528684/fcrl3_3, rs3792876/slc2F2 and rs2268277/runx1) previously reported to be associated with rheumatoid arthritis (RA). The study population consisted of 950 unrelated Japanese subjects with RA and 507 controls, none of whom had previously been tested for these variants. Only the SNP rs2240340/padi4_94 was modestly associated with RA [allele odds ratio (OR) 1.22, 95% confidence interval (CI) 1.04-1.43, P=0.012]. The most significant association effect was found for genotype contrast between minor and major allele homozygotes (OR 1.53, 95% CI 1.10-2.12, P=0.010). No other SNPs showed a statistically significant association with RA in our population. Meta-analysis of published studies and our new data confirmed a highly significant association between PADI4 gene SNPs and increased risk of RA in East Asian populations (allele fixed-effects summary OR 1.31, 95% CI 1.22-1.41, P<0.0001). We found some evidence for an association of either rs7528684/fcrl3_3 or rs3792876/slc2F2 with RA; however, because the magnitudes of effects were apparently much weaker than those reported in the initial positive reports, and there were substantial levels of inter-study OR heterogeneity, we concluded that additional studies are needed to fully understand the present results.
    Journal of Human Genetics 01/2008; 53(2):163-73. · 2.37 Impact Factor
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    ABSTRACT: PKC412 (CGP41251) is a multitarget protein kinase inhibitor with anti-tumor activities. Here, we investigated the effects of PKC412 on macrophages. PKC412 inhibited the proliferation of murine RAW 264.7 macrophages through induction of G2/M cell cycle arrest and apoptosis. At non-toxic drug concentrations, PKC412 significantly suppressed the lipopolysaccharide (LPS)-induced release of TNF-alpha and nitric oxide, while instead enhancing IL-6 secretion. PKC412 attenuated LPS-induced phosphorylations of MKK4 and JNK, as well as AP-1 DNA binding activities. Furthermore, PKC412 suppressed LPS-induced Akt and GSK-3beta phosphorylations. These results suggest that the anti-proliferative and immunomodulatory effects of PKC412 are, at least in part, mediated through its interference with the MKK4/JNK/AP-1 and/or Akt/GSK-3beta pathways. Since macrophages contribute significantly to the development of both acute and chronic inflammation, PKC412 may have therapeutic potential and applications in treating inflammatory and/or autoimmune diseases.
    Biochemical and Biophysical Research Communications 09/2007; 360(1):115-21. · 2.28 Impact Factor
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    ABSTRACT: We have identified novel over-represented and conserved motifs in the upstream regions of human and mouse miRNA stem-loop sequences by means of a new bioinformatic processing regimen. We observed sequence conservation -500 bp upstream in 189 human and mouse miRNAs declining with increasing distance from their putative miRNA stem-loop origin. We also found relatively GC-rich regions having more than 50% of guanine+cytosine (G+C) content at about -30 and -170 bp relative to human miRNA stem-loop sequence origin. To further identify specific sequence motifs that might be involved in the transcriptional regulation of miRNA precursors, we first searched 500 bp upstream sequences of 194 non-redundant human miRNA stem-loop sequences for frequently occurring motifs 5-15 bp long. We then found the comparable occurrences of the 20 most frequent motifs in the 2000 bp upstream regions of 242 human and 290 mouse miRNAs. The significantly reduced frequency of occurrence of all 20 motifs in the regions 2000 bp upstream of 23,570 human RefSeq genes demonstrated that these motifs were specific to the upstream miRNA sequences. The most frequently observed motif M1 (GTGCTTMTAGTGCAG), with a MEME E-value of 3.8e-57 was distributed within 500 bp upstream of stem-loop sequences and was also miRNA-specific. We suggest that these over-represented motif sites are good candidates for experimentally testing miRNA expression as well as possible interaction with regulatory factors.
    Computational Biology and Chemistry 07/2007; 31(3):207-14. · 1.79 Impact Factor
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    ABSTRACT: The human GDD1/TMEM16E gene has been found to be mutated in gnathodiaphyseal dysplasia, an unusual skeletal syndrome with autosomal dominant inheritance. The molecular and biochemical function(s) of GDD1 protein has not yet been elucidated. In this study, we examined the murine GDD1 gene expression pattern during embryonic development, and characterized the cellular and tissue localizations of its gene product using a GDD1-specific antibody. In the developing embryos, GDD1 mRNA expression was principally associated with differentiating and developing somites, with a highly complex spatiotemporal pattern that involved the myotomal and sclerotomal lineages of somites. Biochemical studies indicated that GDD1 protein is an integral membrane glycoprotein that resides predominantly in intracellular vesicles. Immunohistochemical analysis showed a high level of murine GDD1 protein expression in cardiac and skeletal muscle tissues, and in growth-plate chondrocytes and osteoblasts in bone. These observations suggest diverse cellular role(s) of GDD1 in the development of musculoskeletal system.
    Biochemical and Biophysical Research Communications 06/2007; 357(1):126-32. · 2.28 Impact Factor
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    ABSTRACT: Genome-wide maps of linkage disequilibrium (LD) and haplotypes have been created for different populations. Substantial sharing of the boundaries and haplotypes among populations was observed, but haplotype variations have also been reported across populations. Conflicting observations on the extent and distribution of haplotypes require careful examination. The mechanisms that shape haplotypes have not been fully explored, although the effect of sample size has been implicated. We present a close examination of the effect of sample size on haplotype blocks using an original computational simulation. A region spanning 19.31 Mb on chromosome 20q was genotyped for 1,147 SNPs in 725 Japanese subjects. One region of 445 kb exhibiting a single strong LD value (average |D'|; 0.94) was selected for the analysis of sample size effect on haplotype structure. Three different block definitions (recombination-based, LD-based, and diversity-based) were exploited to create simulations for block identification with theta value from real genotyping data. As a result, it was quite difficult to estimate a haplotype block for data with less than 200 samples. Attainment of a reliable haplotype structure with 50 samples was not possible, although the simulation was repeated 10,000 times. These analyses underscored the difficulties of estimating haplotype blocks. To acquire a reliable result, it would be necessary to increase sample size more than 725 and to repeat the simulation 3,000 times. Even in one genomic region showing a high LD value, the haplotype block might be fragile. We emphasize the importance of applying careful confidence measures when using the estimated haplotype structure in biomedical research.
    BMC Bioinformatics 02/2007; 8:200. · 3.02 Impact Factor

Publication Stats

591 Citations
128.49 Total Impact Points

Institutions

  • 2013
    • Kanagawa Children's Medical Center
      Yokohama, Kanagawa, Japan
  • 2004–2013
    • The University of Tokushima
      • • Department of Genetic Information
      • • Diabetes Therapeutics and Research Center
      • • Department of Oral and Maxillofacial Surgery
      Tokusima, Tokushima, Japan
  • 2007
    • Fujitsu Ltd.
      Kawasaki Si, Kanagawa, Japan