Identification of protease serine S1 family member 53 as a mitochondrial protein in murine islet beta cells


Association Study of Fat-mass and Obesity-associated Gene and Body Mass Index in Japanese Patients with Schizophrenia and Healthy Subjects.

Fat-mass and obesity-associated (FTO) gene is known to be involved in the pathophysiology of obesity and a single-nucleotide polymorphism (SNP) rs9939609 of FTO gene is repeatedly confirmed to be associated with body mass index (BMI) and obesity. The aim of this study is to elucidate effects of FTO gene polymorphism on BMI in Japanese patients with schizophrenia and healthy subjects. Three hundred fifty one patients with schizophrenia and 342 age- and sex-matched healthy subjects participated in the study. Information on BMI and antipsychotic medication was also collected from patients and healthy subjects. Genotype of the FTO SNP rs9939609 was determined by TaqMan SNP Genotyping Assays. There was no significant difference in BMI between patients and healthy subjects. No significant difference in BMI was observed among any medications. We observed no significant difference in rs9939609 allele frequencies between patients and healthy subjects. There was a significant difference in BMI between healthy subjects with risk (AA or TA) genotypes and those with TT genotype. We also observed a significant positive correlation between the number of risk allele (A allele) and BMI in healthy subjects. Our study suggested that FTO rs9939609 polymorphism might have some impacts on the BMI in healthy subjects, but might not have same impacts on the BMI of patients with schizophrenia.

Clinical Psychopharmacology and Neuroscience


Diabetic modifier QTL, Dbm4, affecting elevated fasting blood glucose concentrations in congenic mice.

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.


Meta-analysis of association studies between DISC1 missense variants and schizophrenia in the Japanese population.


Bhlhe40, a potential diabetic modifier gene on Dbm1 locus, negatively controls myocyte fatty acid oxidation.

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.


Identification of a novel mutation in the exon 2 splice donor site of the POU1F1/PIT-1 gene in Japanese identical twins with mild combined pituitary hormone deficiency.

Context: 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. Objectives: This study aimed to identify the molecular basis and clinical characteristics of a Japanese CPHD family with a novel POU1F1 mutation. Design: 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. Results: 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 of its transactivation activities in HEK293T cells, while acting as a dominant-negative inhibitor of the endogenous activities of POU1F1 in pituitary GH3 cells. Conclusions: 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.


Analysis of expression and structure of the rat GH-secretagogue/ghrelin receptor (Ghsr) gene: Roles of epigenetic modifications in transcriptional regulation.

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.


Identification and functional analysis of novel human growth hormone secretagogue receptor (GHSR) gene mutations in Japanese subjects with short stature.

Our results suggest that GHSR1A mutations contribute to the genetic etiology of SS in the Japanese population.

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).

The Journal of Clinical Endocrinology and Metabolism


Recessive mutations in the putative calcium-activated chloride channel Anoctamin 5 cause proximal LGMD2L and distal MMD3 muscular dystrophies.

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.


Diabetes mellitus in a Japanese girl with HDR syndrome and GATA3 mutation.

We report on a Japanese girl with HDR (hypoparathyroidism, sensorineural deafness, and renal dysplasia) syndrome who developed diabetes mellitus (DM) at three years of age (blood glucose 713 mg/dL, HbA(1c) 8.0%) in the absence of anti-glutamic acid decarboxylase autoantibodies. Mutation analysis revealed a de novo heterozygous two base pair deletion at exon 6 of the GATA3 gene (c.1200_1201delCA; p.H400fsX506). GATA3 expression was identified by PCR amplification for human pancreas cDNA, and mouse Gata3 was weekly but unequivocally expressed in pancreatic beta cells. The results, in conjunction with the previous findings indicating the critical role of GATA3 in lymphocyte function, GATA3 haploinsufficiency may affect the function of beta cells and/or lymphocytes, leading to the development of DM in relatively exceptional patients with high susceptibility to DM.


Association Study Between the Pericentrin (PCNT) Gene and Schizophrenia.

Disrupted-in-schizophrenia 1 (DISC1), a known genetic risk factor for schizophrenia (SZ) and major depressive disorder (MDD), interacts with several proteins and some of them are reported to be genetically associated with SZ. Pericentrin (PCNT) also interacts with DISC1 and recently single-nucleotide polymorphisms (SNPs) within the PCNT gene have been found to show significant associations with SZ and MDD. In this study, case-controlled association analysis was performed to determine if the PCNT gene is implicated in SZ. Nine SNPs were analyzed in 1,477 individuals (726 patients with SZ and 751 healthy controls). No significant difference was observed between the controls and the patients in allelic frequencies or genotypic distributions of eight SNPs. Although allelic distribution of rs11702684 was different between the two groups (P = 0.042), the difference did not reach statistical significance after permutation correction for multiple comparisons. In the haplotypic analysis, we could not find any significant association in our subjects, either. This gene may not play a major role independently in the etiology of SZ in the Japanese population.


YKL-40 secreted from adipose tissue inhibits degradation of type I collagen.

Obesity is considered a chronic low-grade inflammatory status and the stromal vascular fraction (SVF) cells of adipose tissue (AT) are considered a source of inflammation-related molecules. We identified YKL-40 as a major protein secreted from SVF cells in human visceral AT. YKL-40 expression levels in SVF cells from visceral AT were higher than in those from subcutaneous AT. Immunofluorescence staining revealed that YKL-40 was exclusively expressed in macrophages among SVF cells. YKL-40 purified from SVF cells inhibited the degradation of type I collagen, a major extracellular matrix of AT, by matrix metalloproteinase (MMP)-1 and increased rate of fibril formation of type I collagen. The expression of MMP-1 in preadipocytes and macrophages was enhanced by interaction between these cells. These results suggest that macrophage/preadipocyte interaction enhances degradation of type I collagen in AT, meanwhile, YKL-40 secreted from macrophages infiltrating into AT inhibits the type I collagen degradation.

Biochemical and Biophysical Research Communications


No association between Rho-associated coiled-coil forming protein serine/threonine kinase1 gene and schizophrenia in the Japanese population.


Gene expression and association analyses of the phosphodiesterase 4B (PDE4B) gene in major depressive disorder in the Japanese population.

The phosphodiesterase 4B (PDE4B) interacts with disrupted-in-schizophrenia 1 (DISC1), which is a known genetic risk factor for schizophrenia, bipolar disorder and major depressive disorder (MDD). PDE4B is also important in the regulation of cAMP signaling, a second messenger implicated in learning, memory, and mood. In this study, we determined mRNA expression levels of the PDE4B gene in the peripheral blood leukocytes of patients with MDD and control subjects (n = 33, each). Next we performed two-stage case-controlled association analyses (first set; case = 174, controls = 348; second set; case = 481, controls = 812) in the Japanese population to determine if the PDE4B gene is implicated in MDD. In the leukocytes, a significantly higher expression of the PDE4B mRNA was observed in the drug-naïve MDD patients compared with control subjects (P < 0.0001) and the expression of the MDD patients significantly decreased after antidepressant treatment (P = 0.030). In the association analysis, we observed significant allelic associations of four SNPs (the most significant, rs472952; P = 0.002) and a significant haplotypic association (permutation P = 0.019) between the PDE4B gene and MDD in the first-set samples. However, we could not confirm these significant associations in the following independent second-set of samples. Our results suggest that the PDE4B gene itself does not link to MDD but the elevated mRNA levels of PDE4B might be implicated in the pathophysiology of MDD.

American Journal of Medical Genetics. Part B, Neuropsychiatric Genetics


Positive association of the pericentrin (PCNT) gene with major depressive disorder in the Japanese population.

BACKGROUND: Pericentrin (PCNT) interacts with disruption-in-schizophrenia 1 (DISC1), a known genetic risk factor for schizophrenia, bipolar disorder and major depressive disorder (MDD). We sought to determine whether the PCNT gene is implicated in MDD. METHODS: We performed case-control association analyses in the Japanese population. We analyzed 9 single nucleotide polymorphisms (SNPs) in 173 patients with MDD and 348 healthy controls. RESULTS: We found a significant allelic association between 3 SNPs (rs3788265, rs2073376 and rs2073380) of the PCNT gene and MDD (p = 0.006, 0.005 and 0.021, respectively). After correction for multiple testing, 2 SNPs (rs3788265 and rs2073376) retained significant allelic associations with MDD. In addition, we found a significant association between the 2 marker haplotypes (r3788265 and rs2073376) and MDD (permutation p = 0.011). LIMITATIONS: Our sample was small and comprised only Japanese participants. In addition, owing to the late onset of MDD, it is possible that the disorder will develop in at least some participants in our control group. Finally, we did not show how SNPs of the PCNT gene alter its function. CONCLUSION: Our results suggest that genetic variations in the PCNT gene may play a significant role in the etiology of MDD in the Japanese population.


Association study of TRAF1-C5 polymorphisms with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in Japanese.

The primary aim of this study was to investigate the association of polymorphisms of TRAF1-C5, a newly identified rheumatoid arthritis (RA) risk locus in Caucasians, with susceptibility to RA and systemic lupus erythematosus (SLE) in Japanese populations. Gene expression levels of TRAF1 and C5 to assess the functional significance of genotypes were also analysed. A multicentre association study consisting of 4 RA case-control series (4397 cases and 2857 controls) and 3 SLE case-control series (591 cases and 2199 shared controls) was conducted. Genotyping was performed using TaqMan genotyping assay for two single nucleotide polymorphisms (SNPs) that showed the best evidence of association in the previous Caucasian studies. Quantifications of TRAF1 and C5 expression were performed with TaqMan expression assay. Significant differences in allele frequency for both SNPs were observed between RA and control subjects (combined odds ratio = 1.09), while no significant difference was detected between patients with SLE and controls. Interestingly, alleles rs3761847 A and rs10818488 G had increased the risk for RA in the present study, while they decreased the risk in the original studies. A significant difference was found between risk allele carriers and non-carriers of rs10818488 for the expression level of TRAF1 in phorbol myristate acetate-stimulated lymphoblastoid cell lines (p = 0.04). Association of TRAF1-C5 locus with RA susceptibility was detected in the Japanese populations with modest magnitude, while no significant association was observed for SLE. Significant positive effect of genotype on the expression of TRAF1 might support the genetic association between TRAF1 and RA.


Fragile X carrier screening and FMR1 allele distribution in the Japanese population.

Fragile X syndrome (FXS), which is the most common form of familial mental retardation, is caused by the expansion of the CGG repeat in the FMR1 gene on the X chromosome. Previous studies have suggested that as compared to other populations, Japanese have a lower prevalence of FXS. In addition, in the normal population, there are no carriers who have the premutation allele. We analyzed a total of 946 normal Japanese (576 males and 370 females) and attempted to estimate the frequency of the FMR1 allele. Within this population, we found that 1,155 alleles were in the normal range (less than 40 CGG repeats) and had a modal number of 27 repeats (35.75%). No carriers with premutations (55-200 CGG repeats) were observed in this normal population. We also identified six intermediate-sized alleles (40-54 CGG repeats), with a reported incidence of 1 in 103 males and 1 in 324 females. However, this allele frequency was different from that previously reported for the Japanese population. Since data from previous studies has suggested that FXS might possibly be associated with the genetic mechanism of autism, we also analyzed the length of the CGG repeats in 109 autistic patients. In all cases the CGG repeat numbers were within the normal range (16-36 repeats) and no individuals presented with expanded premutation or intermediate alleles. This finding indicates that the length of the CGG repeat within the FMR1 is unlikely to be responsible for autism in Japanese.


Overexpression of HMGA2 relates to reduction of the let-7 and its relationship to clinicopathological features in pituitary adenomas.

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.


Common coding variant in the TCF7L2 gene and study of the association with type 2 diabetes in Japanese subjects

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.


Altered cellular immunity in transgenic mice with T cell-specific expression of human D4-guanine diphosphate-dissociation inhibitor (D4-GDI).

D4-GDI, a Rho guanosine diphosphate (GDP) dissociation inhibitor, is preferentially expressed in hematopoietic tissues and binds to a small GTP-binding protein, Rho, and inhibits GDP dissociation from Rho. We identified point mutations in the D4-GDI gene in human leukemic cells. We therefore investigated the functions of D4-GDI and mutated D4-GDI in T cells. Transgenic mice (Tg) harboring human wild-type and mutant D4-GDI transgenes driven by the lck promoter were generated. Cellular immunity responses against cytozoic pathogens were examined. The cytoskeletal organization in the CD3+T cells and the proliferation of splenocytes by Con A were investigated in both Tg and littermates (LMs). Granuloma formation by bacille Calmette-Guerin was impaired in the wild-type D4-GDI Tg. On the other hand, the number of granulomas of the mutated D4-Tg was significantly higher. Infection with Listeria was more rapidly fatal to wild-type D4-GDI Tg than to LMs, while the survival of mutated D4-GDI Tg was prolonged. The CD3+T cells in wild-type D4-GDI Tg showed an impairment in the formation of stress fibers on anti-CD3 antibody-coated plates, whereas the cytoskeletal organization in CD3+T cells of the mutated D4-GDI Tg was augmented. The proliferation of splenocytes after Con A stimulation was higher in the mutated D4-GDI Tg than in the LMs. D4-GDI may have important functions, such as induction of T cell migration, adhesion and/or proliferation in inflammatory foci, in cellular immunity responses to cytozoic pathogens.


Transgenic expression of a mutated cyclin-dependent kinase 4 (CDK4/R24C) in pancreatic beta-cells prevents progression of diabetes in db/db mice.

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.


Variants in KCNQ1 are associated with susceptibility to type 2 diabetes mellitus.

We carried out a multistage genome-wide association study of type 2 diabetes mellitus in Japanese individuals, with a total of 1,612 cases and 1,424 controls and 100,000 SNPs. The most significant association was obtained with SNPs in KCNQ1, and dense mapping within the gene revealed that rs2237892 in intron 15 showed the lowest Pvalue (6.7 x 10(-13), odds ratio (OR) = 1.49). The association of KCNQ1 with type 2 diabetes was replicated in populations of Korean, Chinese and European ancestry as well as in two independent Japanese populations, and meta-analysis with a total of 19,930 individuals (9,569 cases and 10,361 controls) yielded a P value of 1.7 x 10(-42) (OR = 1.40; 95% CI = 1.34-1.47) for rs2237892. Among control subjects, the risk allele of this polymorphism was associated with impairment of insulin secretion according to the homeostasis model assessment of beta-cell function or the corrected insulin response. Our data thus implicate KCNQ1 as a diabetes susceptibility gene in groups of different ancestries.


Association of STAT4 with susceptibility to rheumatoid arthritis and systemic lupus erythematosus in the Japanese population.

OBJECTIVE: 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. METHODS: 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). RESULTS: 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. CONCLUSION: 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.


Lack of association of genetic variation in chromosome region 15q14-22.1 with type 2 diabetes in a Japanese population.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.


Positive association of the PDE4B (phosphodiesterase 4B) gene with schizophrenia in the Japanese population.

The phosphodiesterase 4B (PDE4B) gene is located at 1p31, a susceptibility region for schizophrenia (SZ). Moreover, PDE4B interacts with DISC1, which is a known genetic risk factor for SZ. Recently, it was reported that the PDE4B gene is associated with SZ in Caucasian and African American populations. In this study, case-controlled association analyses were performed in the Japanese population to determine if the PDE4B gene is implicated in SZ. Thirteen single nucleotide polymorphisms (SNPs) were analyzed in 444 schizophrenic patients and 452 control subjects. Three SNPs (rs2180335, rs910694 and rs472952) were significantly associated with SZ after applying multiple test correction (p=0.039, 0.004 and 0.028). In addition, a significant association was found between specific haplotypes (rs2180335 and rs910694) and SZ (permutation p=0.001). Our result suggests that variations at the PDE4B locus may play a significant role in the etiology of SZ in the Japanese population.


No association between the NDE1 gene and schizophrenia in the Japanese population.


Single nucleotide polymorphisms in genes encoding LKB1 (STK11), TORC2 (CRTC2) and AMPK alpha2-subunit (PRKAA2) and risk of type 2 diabetes.

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.


The association of genetic variants in Krüppel-like factor 11 and Type 2 diabetes in the Japanese population.

Krüppel-like factor 11 (KLF11) is a transcriptional factor of the zinc finger domain family that regulates the expression of insulin. In North European populations, its common functional variant Q62R (rs35927125) is a strong genetic factor for Type 2 diabetes (P = 0.00033, odds ratio for G allele = 1.29, 95% CI 1.12-1.49). We examined the contribution of KLF11 variants to the susceptibility to Type 2 diabetes in a Japanese population. By re-sequencing Japanese individuals (n = 24, partly 96), we screened all four exons, exon/intron boundaries and flanking regions of KLF11. Verified single nucleotide polymorphisms (SNPs) were genotyped in 731 initial samples (369 control and 362 case subjects). Subsequently, we tested for association in 1087 samples (524 control and 563 case subjects), which were collected in different districts of Japan from the initial samples. We identified eight variants, including a novel A/C variant on intron 3, but no mis-sense mutations. In an association study, we failed to find any significant result of SNPs (minor allele frequency 8.2-46.2%) after correcting for multiple testing. Similarly, no haplotypes were associated with Type 2 diabetes. It is notable that the G allele in rs35927125 was completely absent in 1818 Japanese individuals. Genetic variants in KLF11 are unlikely to have a major effect of Type 2 diabetes in the Japanese population, although they were significantly associated in North European populations. These observations might help to determine the role of KLF11 variants in Type 2 diabetes in different populations.


Diabetic modifier QTLs in F(2) intercrosses carrying homozygous transgene of TGF-beta.

When the homozygous active form of porcine TGF-beta1 transgene (Tgf/Tgf) (under control of the rat glucagon promoter) is introduced into the nonobese diabetic mouse (NOD) genetic background, the mice develop endocrine and exocrine pancreatic hypoplasia, low serum insulin concentrations, and impaired glucose tolerance. To identify genetic modifiers of the diabetic phenotypes, we crossed hemizygous NOD-Tgf with DBA/2J mice (D2) or C3H/HeJ mice (C3H) and used the "transgenic mice" for quantitative trait loci (QTL) analysis. Genome-wide scans of F(2)-D Tgf/Tgf (D2 x NOD) and F(2)-C Tgf/Tgf (C3H x NOD), homozygous for the TGF-beta1 transgene, identified six statistically significant modifier QTLs: one QTL (Tdn1) in F(2)-D Tgf/Tgf, and five QTLs (Tcn1 to Tcn5) in F(2)-C Tgf/Tgf. Tdn1 (Chr 13, LOD = 4.39), and Tcn3 (Chr 2, LOD = 4.94) showed linkage to body weight at 8 weeks of age. Tcn2 (Chr 7, LOD = 4.38) and Tcn4 (Chr 14, LOD = 3.99 and 3.78) showed linkage to blood glucose (BG) concentrations in ipGTT at 30, 0, and 120 min, respectively. Tcn1 (Chr 1, LOD = 4.41) and Tcn5 (Chr 18, LOD = 4.99) showed linkage to serum insulin concentrations in ipGTT at 30 min. Tcn2 includes the candidate gene, uncoupling protein 2 (Ucp2), and shows linkage to Ucp2 mRNA levels in the soleus muscle (LOD = 4.90). Identification of six QTLs for diabetes-related traits in F(2)-D Tgf/Tgf and F(2)-C Tgf/Tgf raises the possibility of identifying candidate susceptibility genes and new targets for drug development for human type 2 diabetes.


Replication of reported genetic associations of PADI4, FCRL3, SLC22A4 and RUNX1 genes with rheumatoid arthritis: results of an independent Japanese population and evidence from meta-analysis of East Asian studies.

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.


Development of an automated SNP analysis method using a paramagnetic beads handling robot.

Biological and medical importance of the single nucleotide polymorphism (SNP) has led to development of a wide variety of methods for SNP typing. Aiming for establishing highly reliable and fully automated SNP typing, we have developed the adapter ligation method in combination with the paramagnetic beads handling technology, Magtration(R). The method utilizes sequence specific ligation between the fluorescently labeled adapter and the sample DNAs at the cohesive end produced by a type IIS restriction enzyme. Evaluation of the method using human genomic DNA showed clear discrimination of the three genotypes without ambiguity using the same reaction condition for any SNPs examined. The operations following PCR amplification were automatically performed by the Magtration(R)-based robot that we have previously developed. Multiplex typing of two SNPs in a single reaction by using four fluorescent dyes was successfully preformed at the almost same sensitivity and reliability as the single typing. These results demonstrate that the automated paramagnetic beads handling technology, Magtration(R), is highly adaptable to the automated SNP analysis and that our method best fits to an automated in-house SNP typing for laboratory and medical uses.


SNPs in the KCNJ11-ABCC8 gene locus are associated with type 2 diabetes and blood pressure levels in the Japanese population.

Many genetic association studies support a contribution of genetic variants in the KCNJ11-ABCC8 gene locus to type 2 diabetes (T2D) susceptibility in Caucasians. In non-Caucasian populations, however, there have been only a few association studies, and discordant results were obtained. Herein, we selected a total of 31 SNPs covering a 211.3-kb region of the KCNJ11-ABCC8 locus, characterized the patterns of linkage disequilibrium (LD) and haplotype structure, and performed a case-control association study in a Japanese population consisting of 909 T2D patients and 893 control subjects. We found significant associations between eight SNPs, including the KCNJ11 E23K and ABCC8 S1369A variants, and T2D. These disease-associated SNPs were genetically indistinguishable because of the presence of strong LD, as found previously in Caucasians. For the KCNJ11 E23K variant, the most significant association was obtained under a dominant genetic model (OR 1.32, 95% CI 1.09-1.60, P = 0.004). A meta-analysis of East Asian studies, comprising a total of 3,357 T2D patients (77.4% Japanese) and 2,836 control subjects (77.8% Japanese), confirmed the significant role of the KCNJ11 E23K variant in T2D susceptibility. Furthermore, we found evidence suggesting that the KCNJ11 E23K genotype is independently associated with higher blood-pressure levels.


The effect of transgenic expression of TGF-beta1 on transplanted islet graft survival.

Transgenic mice expressing the active form of porcine TGF-beta1 (NOD- TGF-beta1 Tg) were completely protected from autoimmune diabetes in the NOD genetic background in our previous study. Here, we attempted to determine whether transgenic expression of TGF-beta1 in transplanted islets prevents autoimmune destruction in NOD mice. We transplanted islets to the subcapsular region of the kidney using NOD-TGF-beta1 Tg and NOD mice as donor and recipient or vice versa. Cyclophosphamide was administered twice to streptozocin-induced diabetic females NOD-TGF-beta1 Tg or their female littermates after islet transplantation. All islets grafts of NOD-TGF-beta1 Tg in spontaneously diabetic NOD mice were rejected earlier than those of their littermates. Hyperglycemia was induced in all littermates, but three out of four NOD-TGF-beta1 Tg, which were STZ-induced diabetic female mice, remained normoglycemic in response to the administration of cyclophosphamide after islet transplantation. Our results lack direct evidence for the local paracrine TGF-beta1 to protect the transplanted islet grafts. We observed, however, prolonged survival of NOD islets grafts in diabetic NOD-TGF-beta1 Tg suggesting the protective role of transgenic TGF-beta1 to suppress the autoimmune process in our syngenic transplantation model. We are convinced that this data could help resolve many problems regarding islet transplantation for type 1 diabetes.


PKC412 (CGP41251) modulates the proliferation and lipopolysaccharide-induced inflammatory responses of RAW 264.7 macrophages.

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


The frequency of the G/G genotype of resistin single nucleotide polymorphism at -420 appears to be increased in younger onset type 2 diabetes.

Resistin is an adipocyte-secreted cytokine associated with insulin resistance in mice. We previously reported that the G/G genotype of a resistin single nucleotide polymorphism (SNP) at -420 increases type 2 diabetes susceptibility by enhancing its promoter activity. The aim of the present study was to determine the relevance of SNP -120 in a large number of subjects. We examined 2,610 type 2 diabetic case and 2,502 control subjects. The relation between SNP -420 and the age of type 2 diabetes onset was further analyzed by adding 237 type 2 diabetic subjects with age of onset <or=40 years. When analyzed without considering subject age, the SNP -420 genotype was not associated with type 2 diabetes. Since we reported that the onset of type 2 diabetes was earlier in G/G genotype, we analyzed the data using a trend test for age intervals of 10 years. The frequency of G/G genotype differed among age grades in type 2 diabetes (P = 0.037) and appeared to be higher in younger grades. In type 2 diabetes, G/G genotype was more frequent in subjects aged <40 years than in those aged >or=40 years (G/G vs. C/C, P = 0.003). In a total of 2,430 type 2 diabetic subjects with age of onset <60 years, the trend test showed that the G/G genotype had an increasing linear trend as the age grade of type 2 diabetes onset became younger (P = 0.0379). In control subjects, the frequency of C/G genotype showed an increasing linear trend with increasing age (P = 0.010). The G/G genotype frequency of resistin SNP -420 appears to be increased in younger-onset type 2 diabetic subjects.


Evaluation of sample size effect on the identification of haplotype blocks.

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.


Parafibromin tumor suppressor enhances cell growth in the cells expressing SV40 large T antigen.

Parafibromin (PF) is a 531-amino acid protein encoded by HRPT2, a putative tumor suppressor gene recently implicated in the autosomal-dominant hyperparathyroidism-jaw tumor familial cancer syndrome and sporadic parathyroid carcinoma. To investigate effects of PF's overexpression on cell proliferation, we performed assays in four different cell lines. The transient overexpression of PF inhibited cell growth in HEK293 and NIH3T3 cells, but enhanced cell growth in the SV40 large T antigen-expressing cell lines such as 293FT and COS7 cells. In 293FT cells, PF was found to interact with SV40 large T antigen and its overexpression promoted entry into the S phase, implying that the interaction enhanced progression through the cell cycle. The tumor suppressor protein PF acts as a positive regulator of cell growth similar to an oncoprotein in the presence of SV40 large T antigen.


Identification of specific sequence motifs in the upstream region of 242 human miRNA genes.

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.

本論文では，miRNAステムループ配列におけるmiRNA特異的上流配列モチーフの同定手法を提案する．提案手法は，ヒトmiRNA上流塩基配列からモチーフ検索プログラムにより検出された配列モチーフの出現頻度を，ヒトmiRNA上流塩基配列と比較対照となるマウスmiRNA上流塩基配列，ヒトタンパク質コーディング遺伝子流塩基配列間で比較・検定することにより，そのmiRNA特異性を検討し，既知転写因子結合部位との配列マトリックス比較により，その新規性を検討する．また，提案手法の有効性検証のために，遺伝子データベースを用いた,配列モチーフ同定実験を行った．


Molecular characterization of GDD1/TMEM16E, the gene product responsible for autosomal dominant gnathodiaphyseal dysplasia.

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.


Genetic association between the PRKCH gene encoding protein kinase Ceta isozyme and rheumatoid arthritis in the Japanese population.

Analyses of families with rheumatoid arthritis (RA) have suggested the presence of a putative susceptibility locus on chromosome 14q21-23. This large population-based genetic association study was undertaken to examine this region. A 2-stage case-control association study of 950 unrelated Japanese patients with RA and 950 healthy controls was performed using >400 gene-based common single-nucleotide polymorphisms (SNPs). Multiple SNPs in the PRKCH gene encoding the eta isozyme of protein kinase C (PKCeta) showed significant single-locus disease associations, the most significant being SNP c.427+8134C>T (odds ratio 0.72, 95% confidence interval 0.62-0.83, P = 5.9 x 10(-5)). Each RA-associated SNP was consistently mapped to 3 distinct regions of strong linkage disequilibrium (i.e., linkage disequilibrium or haplotype blocks) in the PRKCH gene locus, suggesting that multiple causal variants influence disease susceptibility. Significant SNPs included a novel common missense polymorphism of the PRKCH gene, V374I (rs2230500), which lies within the ATP-binding site that is highly conserved among PKC superfamily members. In circulating lymphocytes, PRKCH messenger RNA was expressed at higher levels in resting T cells (CD4(+) or CD8(+)) than in B cells (CD19(+)) or monocytes (CD14(+)) and was significantly down-regulated through immune responses. Our results provide evidence of the involvement of PRKCH as a susceptibility gene for RA in the Japanese population. Dysregulation of PKCeta signal transduction pathway(s) may confer increased risk of RA through aberrant T cell-mediated autoimmune responses.


Genetic association of single nucleotide polymorphisms in endonuclease G-like 1 gene with type 2 diabetes in a Japanese population.

In order to identify type 2 diabetes disease susceptibility gene(s) in a Japanese population, we applied a region-wide case-control association test to the 20.4 Mb region between D3S1293 and D3S2319 on chromosome 3p24.3-22.1, supported by linkage to type 2 diabetes and its related traits in Japanese and multiple populations. We performed a two-stage association test using 1,762 Japanese persons with 485 gene-centric, evenly spaced, common single nucleotide polymorphism (SNP) markers with minor allele frequency >0.1. For mouse studies, total RNA was extracted from various organs of BKS.Cg-+Lepr(db)/+Lepr(db) and control mice, and from MIN6, NIH3T3 and C2C12 cell lines. We detected a landmark SNP375 (A/G) (rs2051211, p = 0.000046, odds ratio = 1.33, 95% CI 1.16-1.53) in intron 5 of the endonuclease G-like 1 (ENDOGL1) gene. Systematic dense SNPs approach identified a susceptibility linkage disequilibrium (LD) block of 116.5 kb by |D'|, an LD units map and a critical region of 2.1 kb by r (2) in ENDOGL1. A haplotype-based association test showed that an at-risk haplotype is associated with disease status (p = 0.00001). The expression of ENDOGL1 was rather ubiquitous with relatively abundant expression in the brain and also in a pancreatic islet beta cell line. Mouse Endogl1 expression increased in pancreatic islets of hyperglycaemic BKS.Cg-+Lepr(db)/+Lepr(db) mice compared with that in control mice. Based on the population genetics, fine mapping of LD block and haplotype analysis, we conclude that ENDOGL1 is a candidate disease-susceptibility gene for type 2 diabetes in a Japanese population. Further analysis in a larger sample size is required to substantiate this conclusion.


Expression and distribution of Gpr119 in the pancreatic islets of mice and rats: Predominant localization in pancreatic polypeptide-secreting PP-cells.

The GPR119 was recently shown to be activated by oleoylethanolamide (OEA), a naturally occurring bioactive lipid with hypophagic and anti-obesity effects. In this study, we have cloned and characterized its murine counterpart, Gpr119. The full-length cDNA contained an open reading frame of 1008bp encoding a 335-amino acid protein. The genomic organization of Gpr119 was unique, having a 3'-untranslated second exon that was also involved in an alternative splicing event. Gene expression analyses confirmed its specific expressions in pancreatic islets and two endocrine cell-lines, MIN6 and alphaTC1. Immunohistochemistry and double-immunofluorescence studies using a specific antibody revealed the predominant Gpr119 localization in pancreatic polypeptide (PP)-cells of islets. No definitive evidence of Gpr119-immunoreactivity in adult beta- or alpha-cells was obtained. The Gpr119 mRNA levels were elevated in islets of obese hyperglycemic db/db mice as compared to control islets, suggesting a possible involvement of this receptor in the development of obesity and diabetes.

Biochemical and Biophysical Research Communications


Effect of +36T > C in intron 1 on the glutamine: fructose-6-phosphate amidotransferase 1 gene and its contribution to type 2 diabetes in different populations.

Glutamine: fructose-6-phosphate amidotransferase 1 (GFPT1) acts as a rate-limiting enzyme in the hexosamine biosynthetic pathway, which is an alternative branch of glucose metabolism. To evaluate GFPT1 as a susceptibility gene to type 2 diabetes, we surveyed the polymorphisms related with the gene function of GFPT1 and assessed its contribution to type 2 diabetes with a case-control association study. Screening of the 5'-flanking and all coding regions of GFPT1 revealed eight polymorphisms, one in the 5'-flanking region, one synonymous polymorphism in exon 8, five in introns and one in 3'-UTR, but no mis-sense or non-sense polymorphism. With in silico simulation, a putative promoter region was apparently predicted between 1 kb upstream and 1 kb downstream of the start codon. In this region, +36T>C polymorphism was located on the GC box sequence in intron 1, and its functional effect on promoter activity was confirmed by luciferase reporter assay, introducing a new functional polymorphism of the GFPT1 gene. To examine its association with type 2 diabetes, we analyzed 2,763 Japanese (1,461 controls and 1,302 cases) and 330 Caucasians (190 controls and 140 cases). One possible association of +36T>C was observed in Caucasians, but no association of polymorphisms including +36T>C in intron 1 or haplotypes was observed in Japanese. Although we could not completely rule out a contribution to specific sub-groups or other populations, genetic variation of GFPT1 is unlikely to have a major role in the susceptibility to type 2 diabetes in Japanese.


Diabetic modifier QTLs identified in F2 intercrosses between Akita and A/J mice.

To identify novel genetic modifiers of type 2 diabetes (T2D), we performed quantitative trait loci (QTL) analysis on F(2) progeny of hypoinsulinemic diabetic Akita mice, heterozygous for the Ins2 gene Cys96Tyr mutation, and nondiabetic A/J mice. We generated 625 heterozygous (F(2)-Hetero) and 338 wild-type (F(2)-Wild) mice with regard to the Ins2 mutation in F(2) intercross progeny. We measured quantitative traits, including plasma glucose and insulin concentrations during the intraperitoneal glucose tolerance test (IPGTT), and body weight (BW). We observed three significant QTLs in hypoinsulinemic hyperglycemic male F(2)-Hetero mice, designated Dbm1, Dbm3, and Dbm4 on Chromosomes 6, 14, and 15, respectively. They showed linkage to plasma glucose concentrations, with significant maximum logarithm of odds (LOD) scores of 4.12, 4.17, and 6.17, respectively, all exceeding threshold values by permutation tests. In normoinsulinemic normoglycemic male F(2)-Wild mice, Dbm1 on Chromosome 6 showed linkage to both plasma insulin concentrations and BW, and Dbm2 on Chromosome 11 showed linkage to plasma glucose concentrations only, with LOD scores of 4.52 and 6.32, and 5.78, respectively. Based on these results, we concluded that Dbm1, Dbm2, Dbm3, and Dbm4 represent four major modifier QTLs specifically affecting T2D-related traits and that these diabetic modifier QTLs are conditional on the heterozygous Ins2 gene mutation and sex to exert their modifier functions. Identification of the genes responsible for these QTLs would provide new drug development targets for human T2D.


Association study on chromosome 20q11.21-13.13 locus and its contribution to type 2 diabetes susceptibility in Japanese.

Several linkage studies have predicted that human chromosome 20q is closely related to type 2 diabetes, but there is no clear evidence that certain variant(s) or gene(s) have strong effects on the disease within this region. To examine disease susceptibility variant in Japanese, verified SNPs from the databases, with a minor allele frequency larger than 0.15, were selected at 10-kb intervals across a 19.31-Mb region (20q11.21-13.13), which contained 291 genes, including hepatocyte nuclear factor 4alpha (HNF4alpha). As a result, a total of 1,147 SNPs were genotyped with TaqMan assay using 1,818 Japanese samples. By searching for HNF4alpha as a representative disease-susceptible gene, no variants of HNF4alpha were strongly associated with disease. To identify other genetic variant related with disease, we designed an extensive two-stage association study (725 first and 1,093 second test samples). Although SNP1146 (rs220076) was selected as a landmark within the 19.31 Mb region, the magnitude of the nominal P value (P = 0.0023) was rather weak. Subsequently, a haplotype-based association study showed that two common haplotypes were weakly associated with disease. All of these tests resulted in non-significance after adjusting for Bonferroni's correction and the false discovery rate to control for the impact of multiple testing. Contrary to the initial expectations, we could not conclude that certain SNPs had a major effect on this promising locus within the framework presented here. As a way to extend our observations, we emphasize the importance of a subsequent association study including replication and/or meta-analysis in multiple populations.


Identification of diabetes susceptibility loci in db mice by combined quantitative trait loci analysis and haplotype mapping.

To identify the disease-susceptibility genes of type 2 diabetes, we performed quantitative trait loci (QTL) analysis in F(2) populations generated from a BKS.Cg-m+/+Lepr(db) and C3H/HeJ intercross, taking advantage of genetically determined obesity and diabetes traits associated with the db gene. A genome-wide scan in the F(2) populations divided by sex and db genotypes identified 14 QTLs in total and 3 major QTLs on chromosome (Chr) 3 (LOD 5.78) for fat pad weight, Chr 15 (LOD 6.64) for body weight, and Chr 16 (LOD 8.15) for blood glucose concentrations. A linear-model-based genome scan using interactive covariates allowed us to consider sex- or sex-by db-specific effects of each locus. For the most significant QTL on Chr 16, the high-resolution haplotype comparison between BKS and C3H strains reduced the critical QTL interval from 20 to 4.6 Mb by excluding shared haplotype regions and identified 11 nonsynonymous single-nucleotide polymorphisms in six candidate genes.


The alpha 2 type IX collagen gene tryptophan polymorphism is not associated with rheumatoid arthritis in the Japanese population

The aim of this study was to investigate whether the alpha 2 type IX collagen (COL9A2) polymorphism that introduces tryptophan residue into the collagen triple-helix is a marker of susceptibility to, or severity of, rheumatoid arthritis (RA). The study included 749 Japanese patients with RA. One hundred twenty-four unrelated healthy individuals served as the control subjects. The relationship between the COL9A2 gene polymorphism and clinical manifestations of RA was evaluated. For the number of subjects positive for COL9A2 tryptophan polymorphism, there was no statistically significant difference between RA patients and normal controls. Furthermore, we did not detect any association of COL9A2 tryptophan polymorphism with disease status, least erosive subset, more erosive subset, or mutilating disease. The lack of association of COL9A2 tryptophan polymorphism with RA and the clinical findings in our study implies that the polymorphism may not function as a candidate gene marker for screening RA patients.


Association of single nucleotide polymorphisms in the suppressor of cytokine signaling 2 (SOCS2) gene with type 2 diabetes in the Japanese.

Several previous linkage scans in type 2 diabetes (T2D) families indicated a putative susceptibility locus on chromosome 12q15-q22, while the underlying gene for T2D has not yet been identified. We performed a region-wide association analysis on 12q15-q22, using a dense set of >500 single-nucleotide polymorphisms (SNPs), in 1492 unrelated Japanese individuals enrolled in this study. We identified an association between T2D and a haplotype block spanning 13.6 kb of genomic DNA that includes the entire SOCS2 gene. Evolutionary-based haplotype analysis of haplotype-tagging SNPs followed by a "sliding window" haplotypic analysis indicated SNPs that mapped to the 5' region of the SOCS2gene to be associated with T2D with high statistical significance. The SOCS2 gene was expressed ubiquitously in human and murine tissues, including pancreatic beta-cell lines. Adenovirus-mediated expression of the SOCS2 gene in MIN6 cells or isolated rat islets significantly suppressed glucose-stimulated insulin secretion. Our data indicate that SOCS2 may play a role in susceptibility to T2D in the Japanese.


No evidence for association of ENPP1(PC-1)K121Q variant with risk of Type 2 diabetes in a Japanese population.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1, also known as PC-1) inhibits insulin signal transduction pathway(s). Previous studies have demonstrated the K121Q variant of the ENPP1 gene to have a significant functional role in determining susceptibility to insulin resistance and type 2 diabetes (T2D). To assess whether the K121Q variant has any impact on T2D in Japanese, we undertook an extensive case-control association study using a total of 911 unrelated Japanese T2D patients and 876 control subjects. No significant difference was observed in either genotype distribution (P=0.95) or allele frequency (P=0.83) between T2D and control groups. Notably, the frequency of the ancestral Q121 allele, which is also present in other primates, was quite high in African-Americans, and showed a marked ethnic variation (77.3% in African-Americans, 16.7% in European Americans, 10.5% in Japanese and 4.2% in Han Chinese). Consequently, the pairwise F(ST )value (a classic measure of genetic distance between pairs of population) showed highly significant differentiations between African-American and non-African-American populations (F(ST)>0.3). Our results indicated that the K121Q variant of the ENPP1 gene has very little, if any, impact on T2D susceptibility in Japanese, but may play a role in the inter-ethnic variability in insulin resistance and T2D.


High-throughput DNA typing of HLA-A, -B, -C, and -DRB1 loci by a PCR-SSOP-Luminex method in the Japanese population.

We have developed a new high-throughput, high-resolution genotyping method for the detection of alleles at the human leukocyte antigen (HLA)-A, -B, -C, and -DRB1 loci by combining polymerase chain reaction (PCR) and sequence-specific oligonucleotide probes (SSOPs) protocols with the Luminex 100 xMAP flow cytometry dual-laser system to quantitate fluorescently labeled oligonucleotides attached to color-coded microbeads. In order to detect the HLA alleles with a frequency of more than 0.1% in the Japanese population, we created 48 oligonucleotide probes for the HLA-A locus, 61 for HLA-B, 34 for HLA-C, and 51 for HLA-DRB1. The accuracy of the PCR-SSOP-Luminex method was determined by comparing it to the nucleotide sequencing method after subcloning into the plasmid vector using 150 multinational control samples obtained from the International HLA DNA Exchange University of California Los Angeles. In addition, we performed the PCR-SSOP-Luminex method for HLA allele typing on DNA samples collected from 1,018 Japanese volunteers. Overall, the genotyping method exhibited an accuracy of 85.91% for HLA-A, 85.03% for HLA-B, 97.32% for HLA-C, and 90.67% for HLA-DRB1 using 150 control samples, and 100% for HLA-A and -C, 99.90% for HLA-B, and 99.95% for HLA-DRB1 in 1,018 Japanese samples. The PCR-SSOP-Luminex method provides a simple, accurate, and rapid approach toward multiplex genotyping of HLA alleles to the four-digit or higher level of resolution in the Japanese population. It takes only approximately 5 h from DNA extraction to the definition of HLA four-digit alleles at the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci for 96 samples when handled by a single typist.


Effect of polymer matrix and glycerol on rapid single-strand conformation polymorphism analysis by capillary and microchip electrophoresis for detection of mutations in K-ras gene

We present the rapid single-strand conformation polymorphism (SSCP) analysis by capillary and microchip electrophoresis to detect the mutations in K-ras gene. Parameters that might affect the analysis of mutation in K-ras gene, such as the polymer and the additive in the sieving matrix, have been studied systematically. Under the optimal conditions, the analysis of seven mutants of K-ras gene could be finished within 10 min by capillary electrophoresis (CE). Furthermore, with the wild-type gene as the inner standard, the analysis accuracy of mutations could be improved. In addition, by studying the properties of polymer solutions, the matrix suitable for microchip electrophoresis was found, and the detection of mutations in K-ras gene could be further shortened to 1 min.


Osteoactivin upregulates expression of MMP-3 and MMP-9 in fibroblasts infiltrated into denervated skeletal muscle in mice

In this study, we examined pathophysiological roles of osteoactivin, a functionally unknown type I membrane glycoprotein, in mouse skeletal muscle atrophied by denervation (sciatic neurectomy). Denervation increased the amounts of osteoactivin, vimentin, matrix metalloproteinase-3 (MMP-3), and MMP-9 in mouse gastrocnemius muscle. Interestingly, immunohistochemical analysis revealed that vimentin, MMP-3, and MMP-9 were mainly present in fibroblast-like cells infiltrated into denervated mouse gastrocnemius muscle, whereas osteoactivin was expressed in the sarcolemma of myofibers adjacent to the fibroblast-like cells. On the basis of these findings, we reasoned that osteoactivin in myocytes was involved in activation of the infiltrated fibroblasts. To address this issue, we examined effects of osteoactivin on expression of MMPs in fibroblasts in vitro and in vivo. Overexpression of osteoactivin in NIH-3T3 fibroblasts induced expression of MMP-3, but not in mouse C(2)C(12) myoblasts, indicating that osteoactivin might functionally target fibroblasts. Treatment with recombinant mouse osteoactivin increased the amounts of collagen type I, MMP-3, and MMP-9 in mouse NIH-3T3 fibroblasts. The upregulated expression of these fibroblast marker proteins was significantly inhibited by heparin, but not by an integrin inhibitor, indicating that a heparin-binding motif in the extracellular domain might be an active site of osteoactivin. In osteoactivin-transgenic mice, denervation further enhanced expression of MMP-3 and MMP-9 in fibroblasts infiltrated into gastrocnemius muscle, compared with wild-type mice. Our present results suggest that osteoactivin might function as an activator for fibroblasts infiltrated into denervated skeletal muscles and play an important role in regulating degeneration/regeneration of extracellular matrix.

American Journal of Physiology, Cell Physiology


Identification of a novel splice site mutation of the CSPG2 gene in a Japanese family with Wagner syndrome.

This linkage study confirmed the genetic homogeneity of the Wagner syndrome. CSPG2 encodes versican, a large chondroitin sulfate proteoglycan, which, in vitreous, binds to hyaluronan and link protein and forms large aggregates that are important for maintaining structural integrity. Although the CSPG2 gene has been excluded as a candidate for causing Wagner syndrome, these data emphasize the necessity of further mutational screening in new families and careful functional characterization.

Investigative Ophthalmology & Visual Science


Molecular cloning and characterization of the murine gnathodiaphyseal dysplasia gene GDD1.

Mutations in the GDD1 gene cause gnathodiaphyseal dysplasia, a rare human skeletal syndrome with autosomal dominant inheritance. The biochemical function(s) of GDD1 protein and the molecular pathophysiology of GDD1 mutations leading to GDD have not yet been elucidated. In this study, we characterized the complete cDNA sequence and genomic organization of the mouse GDD1 gene. Analysis of GDD1 mRNA revealed a complex alternative splicing pattern, involving five exons of the GDD1 gene. GDD1 isoforms lacking conserved amino acids at the N-terminus cytoplasmic tails, and with changes in transmembrane topology, are presumably associated with changes in protein functions and subcellular localizations of GDD1. We found GDD1 expression to be up-regulated during the course of myogenic differentiation in the murine pluripotent mesenchymal precursor cell line C2C12, whereas its expression was diminished during osteoblastic differentiation. These observations suggest diverse cellular roles of GDD1 protein.

Biochemical and Biophysical Research Communications


Identification of candidate genes in the type 2 diabetes modifier locus using expression QTL

To identify new genetic determinants relevant to type 2 diabetes (T2D), diabetic F2 progeny were generated by intercrossing F1 mice obtained from a cross of BKS.Cg-Lepr(db)+/+m and DBA/2, and T2D-related phenotypes were measured. In the F2 population, increased susceptibility to diabetes and obesity was observed. We also detected the major quantitative trait loci (QTL) modifying the severity of diabetes on chromosome 9, where peaks of logarithm of odds (LOD) overlapped for three traits. To identify candidate genes in the QTL intervals, we combined "expression QTL" (eQTL), taking mRNA levels as quantitative traits, and "interstrain sequence variations, including cSNPs." As a result, four genes were identified from cosegregation of clinical QTL with eQTL and 13 genes were found from interstrain cSNPs as candidates in the T2D modifier QTL. Our combined approach shows the acceleration of the discovery of candidate genes in the QTL of interest, spanning several megabases.


Association Between Single-Nucleotide Polymorphisms in the SEC8L1 Gene,Which Encodes a Subunit of the Exocyst Complex,and Rheumatoid Arthritis in a Japanese Population

To identify rheumatoid arthritis (RA) susceptibility genes in a Japanese population by conducting a large-scale case-control association analysis and linkage disequilibrium (LD) mapping on chromosome 7q31-34, a candidate susceptibility locus identified in a preliminary genome-wide scan in 53 Japanese families, using single-nucleotide polymorphisms (SNPs). We prepared 728 dense, evenly spaced SNPs with a minor allele frequency >0.15 in each gene locus on chromosome 7q31-34. Using these SNPs, a 2-stage case-control analysis was performed on 760 RA patients (157 men and 603 women) and 806 non-RA controls (189 men and 617 women). Haplotypes and LD mapping results were assessed based on SNP genotypes in 380 controls. Forty-eight SNPs showed allele associations (P < 0.05) in the first set of DNA samples (380 RA cases and 380 non-RA controls; first-stage analysis). For 4 of the SNPs in the SEC8L1 gene, the association was replicated (P < 0.05) in the second, independent set of DNA samples (an additional 380 RA cases and 380 non-RA controls; second-stage analysis). When data from the 2 groups were combined, the most significant allele association was observed with SNP 441, an intronic SNP of the SEC8L1 gene (P = 0.000059). The SEC8L1 SNPs with significant allele associations were all located in a single conserved LD block (block 4). Haplotype analysis revealed the disease-risk (P = 0.0015) and disease-protective (P = 0.0000062) haplotypes. Resequencing of coding exons within block 4 did not identify any nonsynonymous SNPs. Real-time quantitative polymerase chain reaction revealed that SEC8L1 was expressed ubiquitously in human tissues, including fibroblast-like synoviocytes from RA patients. Our locus-wide association and LD analyses identified intronic SNPs and haplotypes in the SEC8L1 gene that are strongly associated with RA. We propose that SEC8L1, which encodes a component of the exocyst complex, is a candidate susceptibility gene for RA in the Japanese population.


Identification of Cis-Element Regulating Expression of the Mouse Fgf10 Gene during Inner Ear Development

Fibroblast growth factor (FGF) signaling is crucial for the induction and growth of the ear, a sensory organ that involves intimate tissue interactions. Here, we report the abnormality of Fgf10 null ear and the identification of a cis-regulatory element directing otic expression of Fgf10. In Fgf10 null inner ears, we found that the initial development of semicircular, vestibular, and cochlear divisions is roughly normal, after which there are abnormalities of semicircular canal/cristae and vestibular development. The mutant semicircular disks remain without canal formation by the perinatal stage. To elucidate regulation of the Fgf10 expression during inner ear development, we isolated a 6.6-kb fragment of its 5'-upstream region and examined its transcriptional activity with transgenic mice, using a lacZ-reporter system. From comparison of the mouse sequences of the 6.6-kb fragment with corresponding sequences of the human and chicken Fgf10, we identified a 0.4-kb enhancer sequence that drives Fgf10 expression in the developing inner ear. The enhancer sequences have motifs for many homeodomain-containing proteins (e.g., Prx, Hox, Nkx), in addition to POU-domain factors (e.g., Brn3), zinc-finger transcription factors (e.g., GATA-binding factors), TCF/LEF-1, and a SMAD-interacting protein. Thus, FGF10 signaling is dispensable for specification of otic compartment identity but is required for hollowing the semicircular disk. Furthermore, the analysis of a putative inner ear enhancer of Fgf10 has disclosed a complicated regulation of Fgf10 during inner ear development by numerous transcription factors and signaling pathways.

線維芽細胞増殖因子10(FGF10)は内耳の形成に関与していることが，ISH法により解析からわかった．FGF10ノックアウトマウスの解析から内耳が正常に形成されていないことがわかった．さらに，FGF10の5'シス調節エレメントをトランスジェニックマウスを用いて解析したところ，内耳のエンハンサーを同定することに成功した．


The linkage disequilibrium maps of three human chromosomes across four populations reflect their demographic history and a common underlying recombination pattern

The extent and patterns of linkage disequilibrium (LD) determine the feasibility of association studies to map genes that underlie complex traits. Here we present a comparison of the patterns of LD across four major human populations (African-American, Caucasian, Chinese, and Japanese) with a high-resolution single-nucleotide polymorphism (SNP) map covering almost the entire length of chromosomes 6, 21, and 22. We constructed metric LD maps formulated such that the units measure the extent of useful LD for association mapping. LD reaches almost twice as far in chromosome 6 as in chromosomes 21 or 22, in agreement with their differences in recombination rates. By all measures used, out-of-Africa populations showed over a third more LD than African-Americans, highlighting the role of the population's demography in shaping the patterns of LD. Despite those differences, the long-range contour of the LD maps is remarkably similar across the four populations, presumably reflecting common localization of recombination hot spots. Our results have practical implications for the rational design and selection of SNPs for disease association studies.


Hypoplasia of endocrine and exocrine pancreas in homozygous transgenic TGF-β1

We generated the homozygous transgenic mice with expression of the active form of TGF-beta1 by the glucagon promoter (homozygous NOD-TGF-beta1). The homozygous NOD-TGF-beta1 showed severe diabetes in 84.6%, impaired glucose tolerance, and low serum insulin levels. The final size of endocrine and whole pancreas decreased, respectively, to 6 and 34%, compared to wild-type mice. The homozygous N(2) backcross to C57BL/6 (B6-TGF-beta1) showed no diabetes, but impaired glucose tolerance and low serum insulin levels. In homozygous NOD-TGF-beta1, the expression of p15(INK4b) was induced by 3.4-fold in pancreatic islets than that in wild-type mice. Based on these, we conclude first that excessive paracrine TGF-beta1 signaling in islets results in endocrine and exocrine pancreatic hypoplasia, second that TGF-beta1decrease the final size of endocrine and exocrine pancreas presumably through regulating cell cycle via p15(INK4b) at least in endocrine pancreas, and third that hypoplastic action of TGF-beta1 of pancreatic islets is independent of the genetic background.


In vivo proliferation of differentiated pancreatic islet beta cells in transgenic mice expressing mutated cyclin-dependent kinase 4

It has previously been hypothesised that highly differentiated endocrine cells do not proliferate or regenerate. However, recent studies have revealed that cyclin-dependent kinase 4 (CDK4) is necessary for the proliferation of pancreatic islet beta cells. The aim of this study was to determine whether activation of CDK4 can potentially be used as a radical treatment for diabetes without malignant transformation. We generated transgenic mice expressing mutant CDK4 under the control of the insulin promoter to examine the effect of activated CDK4 overexpression in the postnatal development of pancreatic islets. In the transgenic mice, total CDK4 protein expression was increased by up to 5-fold, with a concomitant increase in CDK4 activity indicated by the detection of phosphorylated Rb protein in pancreatic islets. Histopathologically, many cells tested positive for proliferating cell nuclear antigen, and pancreatic islets displayed hyperplasia due to the extreme proliferation of beta cells containing a large number of insulin granules. Pancreatic islet alpha, delta and PP cells did not increase. Over an 18-month observation period, the transgenic mice did not develop insulinoma. Levels of expression of GLUT1 and c-myc were comparable to those in the littermates of the transgenic mice. GLUT2 expression was identified in the pancreatic islets of transgenic mice. No significant differences in telomerase activities were detected between transgenic mice and their littermates. Transgenic mice were superior to their littermates in terms of glucose tolerance and insulin secretion in response to the intraperitoneal injection of glucose, and hypoglycaemia was not observed. Activated CDK4 stimulates postnatal pancreatic beta cell proliferation, during which the highly differentiated phenotypes of pancreatic islet beta cells are preserved without malignant transformation.


Central role for aldose reductase pathway in myocardial ischemic injury


Familial juvenile hyperuricemic nephropathy: Detection of mutations in the uromodulin gene in five Japanese families

Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal-dominant disease characterized by hyperuricemia of underexcretion type, gout, and chronic renal failure. We previously reported linkage on chromosome 16p12 in a large Japanese family designated as family 1 in the present study. Recent reports on the discovery of mutations of the uromodulin (UMOD) gene in families with FJHN encouraged us to screen UMOD mutations in Japanese families with FJHN, including family 1. Six unrelated Japanese families with FJHN were examined for mutations of the UMOD gene by direct sequencing. To confirm the results of the mutation screening, parametric linkage analyses were performed using markers in 16p12 region and around other candidate genes of FJHN. Five separate heterozygous mutations (Cys52Trp, Cys135Ser, Cys195Phe, Trp202Ser, and Pro236Leu) were found in five families, including family 1. All mutations were co-segregated with the disease phenotype in all families, except for family 1, in which an individual in the youngest generation was found as a phenocopy by the genetic testing. Revised multipoint linkage analysis showed that the UMOD gene was located in the interval showing logarithm of odds (LOD) score above 6.0. One family carrying no mutation in the UMOD gene showed no linkage to the medullary cystic kidney disease type 1 (MCKD1) locus, the genes of hepatocyte nuclear factor-1beta (HNF-1beta), or urate transporters URAT1 and hUAT. Our results gave an evidence for the mutation of the UMOD gene in the majority of Japanese families with FJHN. Genetic heterogeneity of FJHN was also confirmed. Genetic testing is necessary for definite diagnosis in some cases especially in the young generation.


The novel gene encoding a putative transmembrane protein is mutated in gnathodiaphyseal dysplasia (GDD).

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of the jawbone. By linkage analysis of a large Japanese family with GDD, we previously mapped the GDD locus to chromosome 11p14.3-15.1. In the critical region determined by recombination mapping, we identified a novel gene (GDD1) that encodes a 913-amino-acid protein containing eight putative transmembrane-spanning domains. Two missense mutations (C356R and C356G) of GDD1 were identified in the two families with GDD (the original Japanese family and a new African American family), and both missense mutations occur at the cysteine residue at amino acid 356, which is evolutionarily conserved among human, mouse, zebrafish, fruit fly, and mosquito. Cellular localization to the endoplasmic reticulum suggests a role for GDD1 in the regulation of intracellular calcium homeostasis.


Differentiation Penotypes of Pancreatic Islet β- and α-cells are Closely Related with Homeotic Genes and a Group of Differentially Expressed Genes.

To identify the genes that determine differentiation phenotypes, we compared gene expression of pancreatic islet beta- and alpha-cells, which are derived from the common precursor and secrete insulin and glucagon, respectively. The expression levels of homeotic genes including Hox genes known to determine region specificity in the antero-posterior (AP) body axis, tissue-specific homeobox genes, and other 8,734 genes were compared in a beta- and alpha-cell line of MIN6 and alpha TC1.6. The expression of homeotic genes were surveyed with reverse transcription-polymerase chain reaction (RT-PCR) using degenerate primers corresponding to invariant amino acid sequences within the homeodomain and subsequently with specific primers. Expression of Hoxc6, Hoxc9, Hoxc10, Pdx1, Cdx2, Gbx2, Pax4, and Hlxb9 genes in MIN6 was higher than those in alpha TC1.6, while expression of Hoxa2, Hoxa3, Hoxa5, Hoxa6, Hoxa7, Hoxa9, Hoxa10, Hoxa13, Hoxb3, Hoxb5, Hoxb6, Hoxb13, Hoxb8, and Brain4 genes in alpha TC1.6 was higher than those in MIN6. Out of 8,734 mouse genes screened with high-density mouse cDNA microarrays for MIN6- and alpha TC1.6-derived cDNA, 58 and 25 genes were differentially over- and under-expressed in MIN6, respectively. GLUTag, which is derived from a large bowel tumor and expresses the proglucagon gene, showed a comparatively similar expression profile to that of alpha TC1.6 in both homeotic and other genes analyzed in cDNA microarray. Our results are consistent with the interpretation that not only the tissue-specific homeotic genes, but also Hox genes are related to differentiation phenotypes of pancreatic beta- and alpha-cells rather than their regional specification of the body in vertebrates.


Redox State-Dependent and Sorbitol Accumulation-Independent Diabetic Albuminuria in Mice with Transgene-Derived Human Aldose Reductase and Sorbitol Dehydrogenase Deficiency.

We investigated the role played by sorbitol accumulation in the kidney in the development of diabetic albuminuria. We created mice ( hAR-Tg:SDH null) with transgene-derived human aldose reductase and sorbitol dehydrogenase (SDH) deficiency, and analysed (i). the contribution of accumulated sorbitol to urinary albumin excretion rate, and (ii). the effect of the aldose reductase inhibitor, epalrestat, on the diabetic redox state, including decreased renal reduced glutathione concentrations or increased lactate to pyruvate ratios in the diabetic kidney. Compared to littermates, non-diabetic transgenic mice had a 2.6-fold increase in aldose reductase mRNA. In a diabetic group, aldose reductase mRNA in hAR-Tg mice was 2.7-fold higher than in littermates. In the diabetic and non-diabetic groups, hAR-Tg:SDH null mice had the highest sorbitol content among all four genetic types including hAR-Tg:SDH null, SDH null, hAR-Tg and littermates. The urinary albumin excretion rate in non-diabetic groups was similar in the four genetic types of mouse. In diabetic groups it was greater than in non-diabetic groups, but did not correlate with the sorbitol content among the four genetic types of mouse. When aldose reductase inhibitor and streptozotocin were given simultaneously at 6 weeks of age, epalrestat prevented diabetic increases in urinary albumin excretion rate and completely prevented diabetic decreases in reduced glutathione concentrations and diabetic increases in lactate to pyruvate ratios, even in the presence of transgenic aldose reductase. The degree of diabetic albuminuria in genetically modified mice is dependent on the redox state and independent of polyol accumulation; aldose reductase inhibitor can prevent diabetic albuminuria by normalising diabetic redox changes.


Gene Cloning and Function Analysis of Replication Factor C from Thermococcus kodakaraensis KOD1

Replication factor C (RFC) catalyzes the assembly of circular proliferating cell nuclear antigen (PCNA) clamps around primed DNA, enabling processive synthesis by DNA polymerase. The RFC-like genes, arranged in tandem in the Thermococcus kodakaraensis KOD1 genome, were cloned individually and co-expressed in Escherichia coli cells. T. kodakaraensis KOD1 RFC homologue (Tk-RFC) consists of the small subunit (Tk-RFCS: MW=37.2 kDa) and the large subunit (Tk-RFCL: MW=57.2 kDa). The DNA elongation rate of the family B DNA polymerase from T. kodakaraensis KOD1 (KOD DNA polymerase), which has the highest elongation rate in all thermostable DNA polymerases, was increased about 1.7 times, when T. kodakaraensis KOD1 PCNA (Tk-PCNA) and the Tk-RFC at the equal molar ratio of KOD DNA polymerase were reacted with primed DNA.

Bioscience, Biotechnology, and Biochemistry


Autosomal Dominant Gnathodiaphyseal Dysplasia Maps to Chromosome 11p14.3-15.1

Gnathodiaphyseal dysplasia (GDD) is a syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of jawbones. Although some cases of this syndrome exist in families with autosomal dominant inheritance, the underlying gene has never been identified. We analyzed a large four-generation family with GDD by linkage analysis using genomic DNA from nine affected and six nonaffected family members. A genome-wide search using a set of highly polymorphic microsatellite markers showed evidence for linkage to chromosome 11p14.3-15.1. Two-point linkage analysis of microsatellite markers spanning this locus resulted in a maximum logarithm of odds (LOD) score of 2.70 with a recombination fraction (theta) of 0 at D11S1755, D11S1759, and D11S915, and a maximum LOD score of 3.01 at D11S4114 was obtained in multipoint linkage analysis. Haplotype analysis detected no recombination between GDD and six closely linked markers (D11S928, D11S1755, D11S4114, D11S1759, D11S915, and D11S929) and established the candidate interval of 8.7 cM on chromosome 11p for GDD. Although GDD has been considered to be a variation of osteogenesis imperfecta (MIM 166260), our results indicate that this syndrome is a new and distinct disease entity from other systemic bone diseases. Furthermore, these genetic markers are useful for presymptomatic diagnosis of GDD in some families and for identification of the GDD gene.


Human Pituitary Adenomas Infrequently Contain Inactivation of Retinoblastoma 1 Gene and Activation of Cyclin Dependent Kinase 4 Gene.

Components of cyclinD1/cyclin-dependent kinase 4 (CDK4)/p16INK4a/pRb pathway are the frequent target of many tumor types. We examined the role of retinoblastoma susceptibility gene (RB1) and the CDK4 gene in human pituitary tumorigenesis. For the RB1 gene, pRb expression and loss of heterozygosity (LOH) on 13q in pituitary adenomas were analysed. Immunostaining of pRb revealed lack of expression in 1 of 29 pituitary adenomas. In 4 of 31 pituitary adenomas, allelic imbalances including LOH of RB1 on 13q14 were detected. Three of 4 pituitary adenomas, in which one adenoma lacked pRb expression, had a common LOH region at least from D13S219 on 13q12.3-q13 to D13S265 on 13q31-32. Interphase fluorescence in situ hybridization with a probe of RB1 showed 2 copies of RB1 gene suggesting that mitotic recombination events, not deletion or chromosome loss, led to LOH in the 3 pituitary adenomas analyzed. All 27 exons, intron-exon boundaries, and essential promoter region of RB1 gene were then sequenced in genomic DNA from 4 pituitary adenomas with allelic imbalance on 13q14 including one adenoma without pRb expression and 3 adenomas with pRb expression. Any somatic mutations, insertions, or microdeletions in the RB1 gene were not detected in 4 pituitary adenomas. Methylation sensitive (MS)-polymerase chain reaction (PCR) and bisulfite sequencing analysis revealed hypomethylated status of CpG islands in the promoter region of the RB1 genes of 4 pituitary adenomas. In addition, activating mutations of CDK4 gene, which is a component of cyclinD1/CDK4/p16INK4a/pRb pathway, were not detected in 31 pituitary adenomas. Based on these results, it is concluded that somatic mutations of the RB1 gene or CDK4 gene do not appear to play a major role in pituitary tumorigenesis. This supports the presence of potential tumor suppressor gene(s) on 13q12.3-q13 to 13q31-32 in pituitary adenomas.


GH-Secreting Pituitary Adenomas Infrequently Contain Inactivating Mutations of PRKAR1A and LOH of 17q23-24.

The molecular events leading to the development of GH-secreting pituitary tumours remain largely unknown. Gsalpha (GNAS1) mutations are found in 27-43% of sporadic GH-secreting adenomas in the Caucasian population, but the frequency of GNAS1 mutations in Japanese and Korean acromegalic patients was reported to be lower, 4-9% and 16%, respectively. Other genes responsible for the tumourigenesis of GH-secreting pituitary adenomas have not been detected yet. PRKAR1A, which codes for the RIalpha regulatory subunit of cyclic AMP-dependent protein kinase A (PKA) on 17q23-24, was recently reported to contain inactivating mutations in some Carney complex families, which involved GH-secreting adenomas in about 10%. We re-evaluated the frequency of GNAS1 mutations and investigated PRKAR1A on the hypothesis that it might play a role in the tumourigenesis of GH-secreting adenomas. We analysed exons 8 and 9 of GNAS1 and all exons and the exon-intron boundaries of PRKAR1A with the PCR and by direct sequencing using genomic DNA extracted from 32 GH-secreting pituitary adenomas (30 GH-secreting adenomas, two GH and PRL-secreting adenomas) and 28 corresponding peripheral blood samples, and performed loss of heterozygosity (LOH) analysis of 17q23-24 with four microsatellite markers and intragenic markers of PRKAR1A. Seventeen of 32 (53.1%) tumours showed somatic-activating mutations of GNAS1: 16 (53.3%) of 30 GH-secreting adenomas and one of two GH and PRL-secreting adenomas. Neither inactivating somatic mutations of PRKAR1A nor LOH of 17q23-24 were detected in any of the tumours examined. We reconfirm the important role of activating mutations of GNAS1 in GH-secreting adenomas, and conclude that PRKAR1A does not play a significant role in the tumourigenesis.


Gene Cloning and Polymerase Chain Reaction with Proliferating Cell Nuclear Antigen from Thermococcus kodakaraensis KOD1

The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 'KOD DNA polymerase'. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.