Manabu Kawamoto

Variations in the FTO gene are associated with severe obesity in the Japanese

AbstractVariations in the fat-mass and obesity-associated gene (FTO) are associated with the obesity phenotype in many Caucasian populations. This association with the obesity phenotype is not clear in the Japanese. To investigate the relationship between the FTO gene and obesity in the Japanese, we genotyped single nucleotide polymorphisms (SNPs) in the FTO genes from severely obese subjects [n = 927, body mass index (BMI) ≥ 30 kg/m2] and normal-weight control subjects (n = 1,527, BMI < 25 kg/m2). A case-control association analysis revealed that 15 SNPs, including rs9939609 and rs1121980, in a linkage disequilibrium (LD) block of approximately 50 kb demonstrated significant associations with obesity; rs1558902 was most significantly associated with obesity. P value in additive mode was 0.0000041, and odds ratio (OR) adjusted for age and gender was 1.41 [95% confidential interval (CI) = 1.22–1.62]. Obesity-associated phenotypes, which include the level of plasma glucose, hemoglobin A1c, total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, and blood pressure were not associated with the rs1558902 genotype. Thus, the SNPs in the FTO gene were found to be associated with obesity, i.e., severe obesity, in the Japanese.

IL-23 induces human osteoclastogenesis via IL-17 in vitro , and anti-IL-23 antibody attenuates collagen-induced arthritis in rats

This study demonstrates that IL-23 stimulates the differentiation of human osteoclasts from peripheral blood mononuclear cells (PBMC). Furthermore, in vivo blockade of endogenous IL-23 activity by treatment with anti-IL-23 antibody attenuates collagen-induced arthritis in rats by preventing both inflammation and bone destruction. IL-23 induced human osteoclastogenesis in cultures of PBMC in the absence of osteoblasts or exogenous soluble-receptor activator of NF-kappaB ligand (RANKL). This IL-23-induced osteoclastogenesis was inhibited by osteoprotegerin, anti-IL-17 antibody, and etanercept, suggesting that RANKL, IL-17, and TNF-alpha are involved. In addition, we found the ratio of production levels of IL-17 to those of IFN-gamma from activated human T cells was elevated at 1 to 10 ng/ml IL-23. The inductive effect of IL-17 and the inhibitory effect of IFN-gamma on osteoclastogenesis indicate that the balance of these two cytokines is particularly important. We also demonstrated that IL-23 administered at a later stage significantly reduced paw volume in rats with collagen-induced arthritis, in a dose-dependent manner. Furthermore, anti-IL-23 antibody reduced synovial tissue inflammation and bone destruction in these rats. These findings suggest that IL-23 is important in human osteoclastogenesis and that neutralizing IL-23 after onset of collagen-induced arthritis has therapeutic potential. Thus, controlling IL-23 production and function could be a strategy for preventing inflammation and bone destruction in patients with rheumatoid arthritis.

Excessive Production of IFN-γ in Patients with Systemic Lupus Erythematosus and Its Contribution to Induction of B Lymphocyte Stimulator/B Cell-Activating Factor/TNF Ligand Superfamily-13B

Expression and immunological significance of IFN-γ, a pivotal cytokine in murine lupus, have not been clearly demonstrated in human systemic lupus erythematosus (SLE). In the present study we investigated the expression of IFN-γ in peripheral blood T cells from patients with SLE and its role in the production of the soluble B lymphocyte stimulator (sBLyS). Peripheral blood T cells from patients with SLE expressed significantly larger amounts of IFN-γ in response to stimulation with anti-CD3 mAb plus anti-CD28 mAb than those from normal controls as shown by three analytical methods, including ELISA, flow cytometry, and quantitative RT-PCR. The ratio of IFN-γ-producing T cells to effector memory T cells in CD3+CD4+ and CD3+CD8+ populations in patients with SLE was significantly higher than that of normal controls. The T-box expressed in T cells (T-bet) mRNA/GATA-binding protein-3 (GATA-3) mRNA ratio was significantly higher in patients with SLE than in normal controls. T cell culture supernatants from patients with SLE contained significantly higher sBLyS-inducing activity than normal controls; this was almost completely inhibited by the addition of anti-human IFN-γ mAb. Percentages of BLyS-expressing peripheral blood monocytes in patients with SLE were significantly higher than those of normal controls. Monocytes from patients with SLE produced significantly larger amounts of sBLyS in response to IFN-γ than those from normal controls. Taken together, these data strongly indicate that the overexpression of IFN-γ in peripheral blood T cells contributes to the immunopathogenesis of SLE via the induction of sBLyS by monocytes/macrophages, which would promote B cell activation and maturation.

IFN-γ-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKL

The current study explored our hypothesis that IFN‐γ‐producing human T cells inhibit human osteoclast formation. Activated T cells derived from human PBMC were divided into IFN‐γ‐producing T cells (IFN‐γ+ T cells) and IFN‐γ‐non‐producing T cells (IFN‐γ– T cells). IFN‐γ+ T cells were cultured with human monocytes in the presence of macrophage‐CSF alone. The concentration of soluble receptor activator of NF‐κB ligand (RANKL) and IFN‐γ, and the amount of membrane type RANKL expressed on T cells, were measured by ELISA. In the patients with early rheumatoid arthritis (RA) treated with non‐steroidal anti‐inflammatory drugs alone, CD4+ T cells expressing both IFN‐γ and RANKL were detected by flow cytometry. Surprisingly, IFN‐γ+ T cells, but not IFN‐γ– T cells, induced osteoclastogenesis from monocytes, which was completely inhibited by adding osteoprotegerin and increased by adding anti‐IFN‐γ antibodies. The levels of both soluble and membrane type RANKL were elevated in IFN‐γ+ T cells. The ratio of CD4+ T cells expressing both IFN‐γ and RANKL in total CD4+ T cells from PBMC was elevated in RA patients. Contrary to our hypothesis, IFN‐γ+ human T cells induced osteoclastogenesis through the expression of RANKL, suggesting that Th1 cells play a direct role in bone resorption in Th1 dominant diseases such as RA.

Association of a functional polymorphism in the IRF5 region with systemic sclerosis in a Japanese population

OBJECTIVE Interferon regulatory factor 5, an established susceptibility factor for systemic lupus erythematosus (SLE), plays a role in type I interferon and proinflammatory cytokine induction. A recent study showed association of a functional single-nucleotide polymorphism (SNP) in intron 1 of IRF5, rs2004640, with systemic sclerosis (SSc) in a European French population. We undertook the present study to determine whether IRF5 polymorphisms are also associated with a predisposition to SSc in Japanese. METHODS A case-control association study was performed for rs2004640 as well as for rs10954213 and rs2280714, all of which were previously reported to be associated with SLE, in 281 SSc patients and 477 healthy controls. Patients with SSc complicated by SLE or Sjögrens syndrome were excluded. Association of the rs2280714 genotype with messenger RNA (mRNA) levels of IRF5 and adjacently located transportin 3 (TNPO3) was examined using the GENEVAR database. RESULTS All 3 SNPs were significantly associated with SSc, with the rs2280714 A allele having the strongest association (allele frequency P=0.0012, odds ratio 1.42 [95% confidence interval 1.15-1.75]). Association was preferentially observed in subsets of patients with diffuse cutaneous SSc (dcSSc) and anti-topoisomerase I antibody positivity. Conditional analysis revealed that rs2280714 could account for most of the association of these SNPs, while an additional contribution of rs2004640 was also suggested for dcSSc. The genotype of rs2280714 was strongly associated with IRF5 mRNA expression, while only marginal association was detected with TNPO3 mRNA expression. CONCLUSION Association of IRF5 with SSc was replicated in a Japanese population. Whether the causal SNP is different among populations requires further investigation.

Large-scale single-nucleotide polymorphism (SNP) and haplotype analyses, using dense SNP Maps, of 199 drug-related genes in 752 subjects: the analysis of the association between uncommon SNPs within haplotype blocks and the haplotypes constructed with haplotype-tagging SNPs.

To optimize the strategies for population-based pharmacogenetic studies, we extensively analyzed single-nucleotide polymorphisms (SNPs) and haplotypes in 199 drug-related genes, through use of 4,190 SNPs in 752 control subjects. Drug-related genes, like other genes, have a haplotype-block structure, and a few haplotype-tagging SNPs (htSNPs) could represent most of the major haplotypes constructed with common SNPs in a block. Because our data included 860 uncommon (frequency <0.1) SNPs with frequencies that were accurately estimated, we analyzed the relationship between haplotypes and uncommon SNPs within the blocks (549 SNPs). We inferred haplotype frequencies through use of the data from all htSNPs and one of the uncommon SNPs within a block and calculated four joint probabilities for the haplotypes. We show that, irrespective of the minor-allele frequency of an uncommon SNP, the majority (mean +/- SD frequency 0.943+/-0.117) of the minor alleles were assigned to a single haplotype tagged by htSNPs if the uncommon SNP was within the block. These results support the hypothesis that recombinations occur only infrequently within blocks. The proportion of a single haplotype tagged by htSNPs to which the minor alleles of an uncommon SNP were assigned was positively correlated with the minor-allele frequency when the frequency was <0.03 (P<.000001; n=233 [Spearmans rank correlation coefficient]). The results of simulation studies suggested that haplotype analysis using htSNPs may be useful in the detection of uncommon SNPs associated with phenotypes if the frequencies of the SNPs are higher in affected than in control populations, the SNPs are within the blocks, and the frequencies of the SNPs are >0.03.

Association between obesity and polymorphisms in SEC16B, TMEM18, GNPDA2, BDNF, FAIM2 and MC4R in a Japanese population

There is evidence that the obesity phenotype in the Caucasian populations is associated with variations in several genes, including neuronal growth regulator 1 (NEGR1), SEC16 homolog B (SCE16B), transmembrane protein 18 (TMEM18), ets variant 5 (ETV5), glucosamine-6-phosphate deaminase 2 (GNPDA2), prolactin (PRL), brain-derived neurotrophic factor (BDNF), mitochondrial carrier homolog 2 (MTCH2), Fas apoptotic inhibitory molecule 2 (FAIM2), SH2B adaptor protein 1 (SH2B1), v-maf musculoaponeurotic fibrosarcoma oncogene homolog (MAF), Niemann-Pick disease, type C1 (NPC1), melanocortin 4 receptor (MC4R) and potassium channel tetramerisation domain containing 15 (KCTD15). To investigate the relationship between obesity and these genes in the Japanese population, we genotyped 27 single-nucleotide polymorphisms (SNPs) in 14 genes from obese subjects (n=1129, body mass index (BMI) ⩾30 kg m−2) and normal-weight control subjects (n=1736, BMI <25 kg m−2). The SNP rs10913469 in SEC16B (P=0.000012) and four SNPs (rs2867125, rs6548238, rs4854344 and rs7561317) in the TMEM18 gene (P=0.00015), all of which were in almost absolute linkage disequilibrium, were significantly associated with obesity in the Japanese population. SNPs in GNPDA2, BDNF, FAIM2 and MC4R genes were marginally associated with obesity (P<0.05). Our data suggest that some SNPs identified by genome-wide association studies in the Caucasians also confer susceptibility to obesity in Japanese subjects.

Association of the FAM167A-BLK region with systemic sclerosis.

OBJECTIVE An association of single-nucleotide polymorphisms (SNPs) in the FAM167A (previously referred to as C8orf13)-BLK region with systemic lupus erythematosus (SLE) has been demonstrated in Caucasians and in Asians. Recent studies have shown that many genes, including IRF5, STAT4, and PTPN22, are shared susceptibility genes in multiple autoimmune diseases. We undertook the current study to examine whether the FAM167A-BLK region is also associated with susceptibility to systemic sclerosis (SSc). METHODS Japanese patients with SSc (n = 309) and healthy controls (n = 769) were enrolled in a 2-tiered case-control association study. In tier 1, 124 patients and 412 controls were tested to determine association of 16 tag SNPs encompassing the FAM167A-BLK region with SSc. In tier 2, an additional 185 patients and 357 controls were analyzed for SNP rs13277113. RESULTS Two haplotype blocks that correspond approximately to FAM167A and BLK were observed. In tier 1 of the study, the rs13277113A allele in the BLK block exhibited the most significant association with SSc after correction for multiple testing (permutated P = 0.024). Two SNP haplotypes formed by rs13277113 and the most significant SNP in the FAM167A block did not exhibit stronger association. When samples from tier 1 and tier 2 were combined, the rs13277113A allele was significantly associated with SSc (odds ratio 1.45 [95% confidence interval 1.17-1.79], P = 6.1 x 10(-4)). Association or a tendency toward association of rs13277113A with SSc was observed regardless of a patients autoantibody profile or whether a patient had diffuse cutaneous or limited cutaneous SSc. CONCLUSION Our findings indicate that the rs13277113A allele is associated not only with SLE but also with SSc and that the FAM167A-BLK region is a common genetic risk factor for both SLE and SSc.

Intracellular IL-1α-binding proteins contribute to biological functions of endogenous IL-1α in systemic sclerosis fibroblasts

The aberrant production of precursor IL-1α (pre-IL-1α) in skin fibroblasts that are derived from systemic sclerosis (SSc) is associated with the induction of IL-6 and procollagen, which contributes to the fibrosis of SSc. However, little is understood about how intracellular pre-IL-1α regulates the expression of the other molecules in fibroblasts. We report here that pre-IL-1α can form a complex with IL-1α-binding proteins that is translocated into the nuclei of fibroblasts. Immunoprecipitation that used anti-human IL-1α Ab and 35S-labeled nuclear extracts of fibroblasts showed three specific bands (≈31, 35, and 65 kDa). The 31-kDa molecule was identified as pre-IL-1α, and the 35- and 65-kDa molecules might be pre-IL-1α-binding proteins. A partial sequencing for the 10 aa from the N-terminals of the molecules showed 100% homology for HAX-1 (HS1-associated protein X-1) and IL-1 receptor type II (IL-1RII). Suppression of the genes of HAX-1 or IL-1RII induced the inhibitory effects of IL-1 signal transduction, including production of IL-6 and procollagen, by fibroblasts. In particular, pre-IL-1α was not translocated into the nucleus by an inhibition of HAX-1. These findings reveal that nuclear localization of pre-IL-1α depends on the binding to HAX-1 and that biological activities might be elicited by the binding to both HAX-1 and IL-1RII in SSc fibroblasts.

Association of STAT4 polymorphism with systemic sclerosis in a Japanese population

Some susceptibility genes are shared by multiple autoimmune diseases. For example, the interferon regulatory factor 5 ( IRF5 ) gene is associated with various autoimmune diseases including systemic lupus erythematosus (SLE) and systemic sclerosis (SSc).1–3 The signal transducer and activator of transcription 4 ( STAT4 ) gene has been shown to be a susceptibility gene for rheumatoid arthritis (RA) and SLE.4 5 STAT4 is a transcription factor involved in interleukin (IL)12 signalling in induction of interferon (IFN)γ production and T helper type 1 (Th1) cell differentiation, type I IFN signalling and IL23-induced IL17 production.6 An association of rs7574864 with SSc was recently reported by a large-scale case-control study in European populations.7 In the present study, we examined the association of a STAT4 intron 3 single nucleotide …