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Whole-genome and -exome resequencing using next-generation sequencers is a powerful approach for identifying genomic variations that are associated with diseases. However, systematic strategies for prioritizing causative variants from many candidates to explain the disease phenotype are still far from being established, because the population-specific frequency spectrum of genetic variation has not been characterized. Here, we have collected exomic genetic variation from 1208 Japanese individuals through a collaborative effort, and aggregated the data into a prevailing catalog. In total, we identified 156 622 previously unreported variants. The allele frequencies for the majority (88.8%) were lower than 0.5% in allele frequency and predicted to be functionally deleterious. In addition, we have constructed a Japanese-specific major allele reference genome by which the number of unique mapping of the short reads in our data has increased 0.045% on average. Our results illustrate the importance of constructing an ethnicity-specific reference genome for identifying rare variants. All the collected data were centralized to a newly developed database to serve as useful resources for exploring pathogenic variations. Public access to the database is available at http://www.genome.med.kyoto-u.ac.jp/SnpDB/.

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.

HTLV-1–associated myelopathy (HAM/TSP) is a chronic and progressive inflammatory disease of the central nervous system. The aim of our study was to identify genetic determinants related to the onset of HAM/TSP in the Japanese population. We conducted a genomewide association study comprising 753 HAM/TSP patients and 899 asymptomatic HTLV-1 carriers. We also performed comprehensive genotyping of HLA-A, -B, -C, -DPB1, -DQB1, and -DRB1 genes using next-generation sequencing technology for 651 HAM/TSP patients and 804 carriers. A strong association was observed in HLA class I (P = 1.54 × 10−9) and class II (P = 1.21 × 10−8) loci with HAM/TSP. Association analysis using HLA genotyping results showed that HLA-C*07:02 (P = 2.61 × 10−5), HLA-B*07:02 (P = 4.97 × 10−10), HLA-DRB1*01:01 (P = 1.15 × 10−9) and HLA-DQB1*05:01 (P = 2.30 × 10−9) were associated with disease risk, while HLA-B*40:06 (P = 3.03 × 10−5), HLA-DRB1*15:01 (P = 1.06 × 10−5) and HLA-DQB1*06:02 (P = 1.78 × 10−6) worked protectively. Logistic regression analysis identified amino acid position 7 in the G-BETA domain of HLA-DRB1 as strongly associated with HAM/TSP (P = 9.52 × 10−10); individuals homozygous for leucine had an associated increased risk of HAM/TSP (odds ratio, 9.57), and proline was protective (odds ratio, 0.65). Both associations were independent of the known risk associated with proviral load. DRB1-GB-7-Leu was not significantly associated with proviral load. We have identified DRB1-GB-7-Leu as a genetic risk factor for HAM/TSP development independent of proviral load. This suggests that the amino acid residue may serve as a specific marker to identify the risk of HAM/TSP even without knowledge of proviral load. In light of its allele frequency worldwide, this biomarker will likely prove useful in HTLV-1 endemic areas across the globe.

Background A previous study revealed the association between susceptibility to Takayasu arteritis (TAK) and a single nucleotide polymorphism (SNP) rs6871626 located in IL12B , which encodes interleukin (IL)-12p40, a common component of IL-12p70 and IL-23. We investigated the expression of these cytokines in patients with TAK, stratifying them into those with or without the risk allele at the rs6871626 SNP. Methods Plasma levels of IL-12p40, IL-12p70, and IL-23 were quantified in 44 patients with TAK and 19 healthy controls (HCs) by enzyme-linked immunosorbent assays. Monocytes were obtained from 20 patients with TAK and 14 HCs, treated with interferon-γ (IFN-γ) and lipopolysaccharide, and then supernatant cytokines were quantified. In addition, the ratio of IFN-γ + or IL-17A + cells to CD4 + T cells was measured by flow cytometric analysis of peripheral blood mononuclear cells. Results The levels of plasma IL-12p40, plasma IL-12p70, and supernatant IL-12p70 were significantly higher in patients with TAK than in HCs, whereas there were no significant differences in the levels of plasma IL-23, supernatant IL-23, or supernatant IL-12p40. The levels of plasma IL-12p70, supernatant IL-12p40, and supernatant IL-12p70 were significantly higher in patients with the risk allele than in those without. The ratio of CD4 + IFN-γ + cells was significantly higher in patients with the risk allele, whereas CD4 + IL-17A + cells showed no differences. Conclusions The rs6871626 SNP in IL12B may influence the increased expression of IL-12p40 and IL-12p70. These enhanced cytokines might play roles in the pathophysiology of TAK.

Background Pulmonary arterial hypertension (PAH) is a severe lung disease with only few effective treatments available. Familial cases of PAH are usually recognized as an autosomal dominant disease, but incomplete penetrance of the disease makes it difficult to identify pathogenic variants in accordance with a Mendelian pattern of inheritance. Methods To elucidate the complex genetic basis of PAH, we obtained whole exome- or genome-sequencing data of 17 subjects from 9 families with heritable PAH and applied gene-based association analysis with 9 index patients and 300 PAH-free controls. Results A burden of rare variants in BMPR2 significantly contributed to the risk of the disease ( p  = 6.0 × 10 −8 ). Eight of nine families carried four previously reported single nucleotide variants and four novel insertion/deletion variants in the gene. One of the novel variants was a large 6.5 kilobase-deletion. In the remaining one family, the patient carried a pathogenic variant in a member of potassium channels, KCNK3, which was the first replicative finding of channelopathy in an Asian population. Conclusions The variety of rare pathogenic variants suggests that gene-based association analysis using genome-wide sequencing data from increased number of samples is essential to tracing the genetic heterogeneity and developing an appropriate panel for genetic testing.

Background Prostate-specific antigen (PSA) is a useful marker for prostate cancer (PCa) and is widely used for screening of PCa. Previous studies have shown that genetic components influence the levels of PSA, and some of these genetic components would lead to better diagnostic sensitivity and specificity to PCa. However, genetic studies for PSA from Asian countries are limited. Our aim was to identify genetic components influencing PSA levels in the Japanese population using genome-wide association study (GWAS) and to analyse whether genetic components would lead to better screening abilities of PCa. Methods We performed a GWAS comprising 1086 male subjects using 303 283 single nucleotide proteins, followed by a replication study of 1302 subjects. PSA levels were quantified by chemiluminescence immunoassay method. Quantitative linear regression analysis was performed to assess genetic components of PSA levels. A total of 413 subjects with prostate biopsies were analysed to examine whether genetic determinants would improve diagnostic ability. Results Rs16856139 in SLC45A3, the same region as the previous Chinese study, showed an overall significant association with PSA levels (p=2.4×10−11) along with rs1058205 in KLK3. In silico analysis revealed significant association between rs16856139 and expression of SLC45A3. Genetic scores of PSA showed a dose-dependent decrease of area under curve (AUC) of PCa and successfully subgrouped the individuals with significantly different AUC (p≤0.0097). Conclusions Rs16856139, associated with the expression of SLC45A3, is significantly associated with the levels of PSA in the Japanese population. Classification of subjects based on PSA genetic determinants would improve screening ability of PSA to detect PCa.

Rheumatoid arthritis (RA) is a major cause of adult chronic inflammatory arthritis and a typical complex trait. Although several genetic determinants have been identified, they account for only a part of the genetic susceptibility. We conducted a genome-wide association study of RA in Japanese using 225,079 SNPs genotyped in 990 cases and 1,236 controls from two independent collections (658 cases and 934 controls in collection1; 332 cases and 302 controls in collection2), followed by replication studies in two additional collections (874 cases and 855 controls in collection3; 1,264 cases and 948 controls in collection4). SNPs showing p<0.005 in the first two collections and p<10(-4) by meta-analysis were further genotyped in the latter two collections. A novel risk variant, rs2000811, in intron2 of the myelin basic protein (MBP) at chromosome 18q23 showed strong association with RA (p = 2.7×10(-8), OR 1.23, 95% CI: 1.14-1.32). The transcription of MBP was significantly elevated with the risk allele compared to the alternative allele (p<0.001). We also established by immunohistochemistry that MBP was expressed in the synovial lining layer of RA patients, the main target of inflammation in the disease. Circulating autoantibody against MBP derived from human brain was quantified by ELISA between patients with RA, other connective tissue diseases and healthy controls. As a result, the titer of anti-MBP antibody was markedly higher in plasma of RA patients compared to healthy controls (p<0.001) and patients with other connective tissue disorders (p<0.001). ELISA experiment using citrullinated recombinant MBP revealed that a large fraction of anti-MBP antibody in RA patients recognized citrullinated MBP. This is the first report of a genetic study in RA implicating MBP as a potential autoantigen and its involvement in pathogenesis of the disease.

Calpain, a calcium-dependent cysteine protease, is reportedly involved in the pathophysiology of autoimmune diseases such as rheumatoid arthritis (RA). In addition, autoantibodies against calpastatin, a natural and specific inhibitor of calpain, are widely observed in RA. We previously reported that E-64-d, a membrane-permeable cysteine protease inhibitor, is effective in treating experimental arthritis. However, the exact role of the calpastatin-calpain balance in primary inflammatory cells remains unclear. Here we investigated the effect of calpain-specific inhibition by overexpressing a minimal functional domain of calpastatin in primary helper T (Th) cells, primary fibroblasts from RA patients, and fibroblast cell lines. We found that the calpastatin-calpain balance varied during Th1, Th2, and Th17 development, and that overexpression of a minimal domain of calpastatin (by retroviral gene transduction) or the inhibition of calpain by E-64-d suppressed the production of IL-6 and IL-17 by Th cells and the production of IL-6 by fibroblasts. These suppressions were associated with reductions in RORγt expression and STAT3 phosphorylation. Furthermore, inhibiting calpain by silencing its small regulatory subunit (CPNS) suppressed Th17 development. We also confirmed that overexpressing a minimal domain of calpastatin suppressed IL-6 by reducing NF-κB signaling via the stabilization of IκBα, without affecting the upstream signal. Moreover, our findings indicated that calpastatin overexpression suppressed IL-17 production by Th cells by up-regulating the STAT5 signal. Finally, overexpression of a minimal domain of calpastatin suppressed IL-6 production efficiently in primary fibroblasts derived from the RA synovium. These findings suggest that inhibiting calpain by overexpressing a minimal domain of calpastatin could coordinately suppress proinflammatory activities, not only those of Th cells but also of synovial fibroblasts. Thus, this strategy may prove viable as a candidate treatment for inflammatory diseases such as RA.

Takayasu arteritis (TAK) is a systemic vasculitis with severe complications that affects the aorta and its large branches. HLA-B*52 is an established susceptibility locus to TAK. To date, there are still only a limited number of reports concerning non-HLA susceptibility loci to TAK. We conducted a genome-wide association study (GWAS) and a follow-up study in a total of 633 TAK cases and 5,928 controls. A total of 510,879 SNPs were genotyped, and 5,875,450 SNPs were imputed together with HLA-B*52. Functional annotation of significant loci, enhancer enrichment, and pathway analyses were conducted. We identified four unreported significant loci, namely rs2322599, rs103294, rs17133698, and rs1713450, in PTK2B, LILRA3/LILRB2, DUSP22, and KLHL33, respectively. Two additional significant loci unreported in non-European GWAS were identified, namely HSPA6/FCGR3A and chr21q.22. We found that a single variant associated with the expression of MICB, a ligand for natural killer (NK) cell receptor, could explain the entire association with the HLA-B region. Rs2322599 is strongly associated with the expression of PTK2B. Rs103294 risk allele in LILRA3/LILRB2 is known to be a tagging SNP for the deletion of LILRA3, a soluble receptor of HLA class I molecules. We found a significant epistasis effect between HLA-B*52 and rs103294 (P = 1.2 × 10−3). Enhancer enrichment analysis and pathway analysis suggested the involvement of NK cells (P = 8.8 × 10−5, enhancer enrichment). In conclusion, four unreported TAK susceptibility loci and an epistasis effect between LILRA3 and HLA-B*52 were identified. HLA and non-HLA regions suggested a critical role for NK cells in TAK.

Introduction Although susceptibility genes for anti-citrullinated peptide/protein antibodies (ACPA)-positive rheumatoid arthritis (RA) have been successfully discovered by genome-wide association studies (GWAS), little is known about the genetic background of ACPA-negative RA. We intended to elucidate genetic background of ACPA-negative RA. Method We performed a meta-analysis of GWAS comprising 670 ACPA-negative RA and 16,891 controls for 1,948,138 markers, followed by a replication study of the top 35 single nucleotide polymorphisms (SNPs) using 916 cases and 3,764 controls. Inverse-variance method was applied to assess overall effects. To assess overlap of susceptibility loci between ACPA-positive and -negative RA, odds ratios (ORs) of the 21 susceptibility markers to RA in Japanese were compared between the two subsets. In addition, SNPs were stratified by the p-values in GWAS meta-analysis for either ACPA-positive RA or ACPA-negative RA to address the question whether weakly-associated genes were also shared. The correlations between ACPA-positive RA and the subpopulations of ACPA-negative RA (rheumatoid factor (RF)-positive and RF-negative subsets) were also addressed. Results Rs6904716 in LEMD2 of the human leukocyte antigen (HLA) locus showed a borderline association with ACPA-negative RA (overall p = 5.7 × 10 −8 ), followed by rs6986423 in CSMD1 (p = 2.4 × 10 −6 ) and rs17727339 in FCRL3 (p = 1.4 × 10 −5 ). ACPA-negative RA showed significant correlations of ORs with ACPA-positive RA for the 21 susceptibility SNPs and non-HLA SNPs with p-values far from significance. These significant correlations with ACPA-positive RA were true for ACPA-negative RF-positive and ACPA-negative RF-negative RA. On the contrary, positive correlations were not observed between the ACPA-negative two subpopulations. Conclusion Many of the susceptibility loci were shared between ACPA-positive and -negative RA.