Chikashi Terao

Genetics of rheumatoid arthritis contributes to biology and drug discovery

A major challenge in human genetics is to devise a systematic strategy to integrate disease-associated variants with diverse genomic and biological data sets to provide insight into disease pathogenesis and guide drug discovery for complex traits such as rheumatoid arthritis (RA). Here we performed a genome-wide association study meta-analysis in a total of >100,000 subjects of European and Asian ancestries (29,880 RA cases and 73,758 controls), by evaluating ∼10 million single-nucleotide polymorphisms. We discovered 42 novel RA risk loci at a genome-wide level of significance, bringing the total to 101 (refs 2, 3, 4). We devised an in silico pipeline using established bioinformatics methods based on functional annotation, cis-acting expression quantitative trait loci and pathway analyses—as well as novel methods based on genetic overlap with human primary immunodeficiency, haematological cancer somatic mutations and knockout mouse phenotypes—to identify 98 biological candidate genes at these 101 risk loci. We demonstrate that these genes are the targets of approved therapies for RA, and further suggest that drugs approved for other indications may be repurposed for the treatment of RA. Together, this comprehensive genetic study sheds light on fundamental genes, pathways and cell types that contribute to RA pathogenesis, and provides empirical evidence that the genetics of RA can provide important information for drug discovery.

Genome-wide association study and gene expression analysis identifies CD84 as a predictor of response to etanercept therapy in rheumatoid arthritis.

Anti-tumor necrosis factor alpha (anti-TNF) biologic therapy is a widely used treatment for rheumatoid arthritis (RA). It is unknown why some RA patients fail to respond adequately to anti-TNF therapy, which limits the development of clinical biomarkers to predict response or new drugs to target refractory cases. To understand the biological basis of response to anti-TNF therapy, we conducted a genome-wide association study (GWAS) meta-analysis of more than 2 million common variants in 2,706 RA patients from 13 different collections. Patients were treated with one of three anti-TNF medications: etanercept (n = 733), infliximab (n = 894), or adalimumab (n = 1,071). We identified a SNP (rs6427528) at the 1q23 locus that was associated with change in disease activity score (ΔDAS) in the etanercept subset of patients (P = 8×10−8), but not in the infliximab or adalimumab subsets (P>0.05). The SNP is predicted to disrupt transcription factor binding site motifs in the 3′ UTR of an immune-related gene, CD84, and the allele associated with better response to etanercept was associated with higher CD84 gene expression in peripheral blood mononuclear cells (P = 1×10−11 in 228 non-RA patients and P = 0.004 in 132 RA patients). Consistent with the genetic findings, higher CD84 gene expression correlated with lower cross-sectional DAS (P = 0.02, n = 210) and showed a non-significant trend for better ΔDAS in a subset of RA patients with gene expression data (n = 31, etanercept-treated). A small, multi-ethnic replication showed a non-significant trend towards an association among etanercept-treated RA patients of Portuguese ancestry (n = 139, P = 0.4), but no association among patients of Japanese ancestry (n = 151, P = 0.8). Our study demonstrates that an allele associated with response to etanercept therapy is also associated with CD84 gene expression, and further that CD84 expression correlates with disease activity. These findings support a model in which CD84 genotypes and/or expression may serve as a useful biomarker for response to etanercept treatment in RA patients of European ancestry.

A Genome-Wide Association Study Identified AFF1 as a Susceptibility Locus for Systemic Lupus Eyrthematosus in Japanese

Systemic lupus erythematosus (SLE) is an autoimmune disease that causes multiple organ damage. Although recent genome-wide association studies (GWAS) have contributed to discovery of SLE susceptibility genes, few studies has been performed in Asian populations. Here, we report a GWAS for SLE examining 891 SLE cases and 3,384 controls and multi-stage replication studies examining 1,387 SLE cases and 28,564 controls in Japanese subjects. Considering that expression quantitative trait loci (eQTLs) have been implicated in genetic risks for autoimmune diseases, we integrated an eQTL study into the results of the GWAS. We observed enrichments of cis-eQTL positive loci among the known SLE susceptibility loci (30.8%) compared to the genome-wide SNPs (6.9%). In addition, we identified a novel association of a variant in the AF4/FMR2 family, member 1 (AFF1) gene at 4q21 with SLE susceptibility (rs340630; P = 8.3×10−9, odds ratio = 1.21). The risk A allele of rs340630 demonstrated a cis-eQTL effect on the AFF1 transcript with enhanced expression levels (P<0.05). As AFF1 transcripts were prominently expressed in CD4+ and CD19+ peripheral blood lymphocytes, up-regulation of AFF1 may cause the abnormality in these lymphocytes, leading to disease onset.

Two Susceptibility Loci to Takayasu Arteritis Reveal a Synergistic Role of the IL12B and HLA-B Regions in a Japanese Population

Takayasu arteritis (TAK) is an autoimmune systemic vasculitis of unknown etiology. Although previous studies have revealed that HLA-B*52:01 has an effect on TAK susceptibility, no other genetic determinants have been established so far. Here, we performed genome scanning of 167 TAK cases and 663 healthy controls via Illumina Infinium Human Exome BeadChip arrays, followed by a replication study consisting of 212 TAK cases and 1,322 controls. As a result, we found that the IL12B region on chromosome 5 (rs6871626, overall p = 1.7 × 10(-13), OR = 1.75, 95% CI 1.42-2.16) and the MLX region on chromosome 17 (rs665268, overall p = 5.2 × 10(-7), OR = 1.50, 95% CI 1.28-1.76) as well as the HLA-B region (rs9263739, a proxy of HLA-B*52:01, overall p = 2.8 × 10(-21), OR = 2.44, 95% CI 2.03-2.93) exhibited significant associations. A significant synergistic effect of rs6871626 and rs9263739 was found with a relative excess risk of 3.45, attributable proportion of 0.58, and synergy index of 3.24 (p ≤ 0.00028) in addition to a suggestive synergistic effect between rs665268 and rs926379 (p ≤ 0.027). We also found that rs6871626 showed a significant association with clinical manifestations of TAK, including increased risk and severity of aortic regurgitation, a representative severe complication of TAK. Detection of these susceptibility loci will provide new insights to the basic mechanisms of TAK pathogenesis. Our findings indicate that IL12B plays a fundamental role on the pathophysiology of TAK in combination with HLA-B(∗)52:01 and that common autoimmune mechanisms underlie the pathology of TAK and other autoimmune disorders such as psoriasis and inflammatory bowel diseases in which IL12B is involved as a genetic predisposing factor.

The human AIRE gene at chromosome 21q22 is a genetic determinant for the predisposition to rheumatoid arthritis in Japanese population

Rheumatoid arthritis (RA) is a typical complex trait and the major cause of chronic inflammation worldwide. Although multiple genetic loci have been shown for their association with the onset of RA, they cover only a part of its genetic components and are largely ethnicity-specific. To identify novel genetic factors related to the predisposition and prognosis of RA in Japanese, we conducted a large-scale genome-wide association (GWA) study. We performed a GWA analysis by scanning the genome of 1247 RA cases and 1486 controls for 277 420 single nucleotide polymorphisms (SNPs), followed by replication analysis using two independent sample sets consisting of 1865 cases and 1623 controls, and 2303 cases and 3380 controls. We identified two SNPs, rs2075876 and rs760426, in intron of the autoimmune regulator AIRE gene at chromosome 21q22 that showed strong associations with the disease (P= 3.6 × 10(-9) and P= 4.4 × 10(-8), respectively). Rs1800250, in exon7 of AIRE, was in strong linkage disequilibrium (r(2)= 0.94) with rs2075876 and introduced an amino acid alteration (S278R) in the SAND domain of the AIRE protein. In silico analysis showed the decreased transcription of AIRE by the risk allele of rs2075876 compared with the alternative allele (P= 6.8 × 10(-5)). No correlation was observed between the rs2075876 genotype and quantitative traits reflecting the progression of RA. As AIRE is a key molecule which regulates the expression and presentation of self-antigens in thymic negative selection, its downregulation by genetic polymorphisms may result in the survival of auto-reactive T cells to trigger auto-inflammation in RA.

Anti-citrullinated peptide antibody-negative RA is a genetically distinct subset: a definitive study using only bone-erosive ACPA-negative rheumatoid arthritis

Objectives. ACPA is a highly specific marker for RA. It was recently reported that ACPA can be used to classify RA into two disease subsets, ACPA-positive and ACPA-negative RA. ACPA-positive RA was found to be associated with the HLA-DR shared epitope (SE), but ACPA negative was not. However, the suspicion remained that this result was caused by the ACPA-negative RA subset containing patients with non-RA diseases. We examined whether this is the case even when possible non-RA ACPA-negative RA patients were excluded by selecting only patients with bone erosion. Methods. We genotyped HLA-DRB1 alleles for 574 ACPA-positive RA, 185 ACPA-negative RA (including 97 erosive RA) and 1508 healthy donors. We also tested whether HLA-DR SE is associated with RF-negative or ANA-negative RA. Results. ACPA-negative RA with apparent bone erosion was not associated with SE, supporting the idea that ACPA-negative RA is genetically distinct from ACPA-positive RA. We also tested whether these subsets are based on autoantibody-producing activity. In accordance with the ACPA-negative RA subset, the RF-negative RA subset showed a clearly distinct pattern of association with SE from the RF-positive RA. In contrast, ANA-negative as well as ANA-positive RA was similarly associated with SE, suggesting that the subsets distinguished by ACPA are not based simply on differences in autoantibody production. Conclusions. ACPA-negative erosive RA is genetically distinct from ACPA-positive RA.

A Clinical, Pathological, and Genetic Characterization of Methotrexate-associated Lymphoproliferative Disorders

Objective. Methotrexate-associated lymphoproliferative disorders (MTX-LPD) often regress spontaneously during MTX withdrawal, but the prognostic factors remain unclear. The aim of our study was to clarify the clinical, histological, and genetic factors that predict outcomes in patients with MTX-LPD. Methods. Patients with MTX-LPD diagnosed between 2000 and 2012 were analyzed retrospectively regarding their clinical course, site of biopsy, histological typing, Epstein-Barr virus (EBV) in situ hybridization and immunostaining, and HLA type. Results. Twenty-one patients, including 20 with rheumatoid arthritis (RA) and 1 with polymyositis, were analyzed. The mean dose of MTX was 6.1 mg/week and the mean duration of treatment was 71.1 months. Clinically, 5 patients were diagnosed with EBV-positive mucocutaneous ulcer (EBVMCU) and had polymorphic histological findings. The proportion of those patients successfully treated solely by withdrawal of MTX was significantly greater than that of those without EBVMCU (75% vs 7.7%, p = 0.015). The HLA-B15:11 haplotype was more frequent in patients with EBV+ RA with MTX-LPD than in healthy Japanese controls (p = 0.0079, Bonferroni’s method). EBV latency classification and HLA typing were not associated with the prognosis of MTX-LPD in our cohort. Conclusion. Our data demonstrate that patients in the EBVMCU, a specific clinical subgroup of MTX-LPD, had a better clinical outcome when MTX was withdrawn than did other patients with MTX-LPD.

Recent advances in Takayasu arteritis.

Takayasu arteritis (TAK) is a relatively rare systemic vasculitis mainly affecting the aorta and its large branches. While patients with TAK are more frequently observed in Asian countries, we can find patients with TAK all over the world. This limited number of patients has made it difficult to collect large numbers of patients and perform detailed studies. However, recent progresses have led to the identification of susceptibility genes and novel susceptibility human leukocyte antigen (HLA) alleles as well as accumulation of clues for the pathophysiology of TAK. IL12B was shown to be a susceptibility gene beyond ethnicity. MLX and FCGR2A/3A were shown to be associated with TAK in Japanese and Turkish/American populations, respectively. HLA‐B*52:01 and *67:01 are susceptibility alleles to TAK, and the 171st and 67th amino acid residues of HLA‐B protein are suggested important for TAK susceptibility. HLA‐DQB1/DRB1 is recently reported as an independent susceptibility locus. Although there are no standardized serum markers or composite measures for disease activity of TAK, Japanese and Italian groups showed pentraxin 3 as a novel biomarker for detecting and monitoring patients with TAK. Recently, an Indian group proposed a novel scoring system called ITAS to evaluate disease activity of TAK. Standardization of assessing disease activity would lead to clinical studies with high quality. Several groups reported results of treatment for refractory TAK with biological agents targeting tumor necrosis factor or interleukin‐6R. The recent accumulation of research data should improve understanding of the basic pathophysiology of TAK and lead to better management of patients with TAK.

Neutrophils Are Essential As A Source Of Il-17 In The Effector Phase Of Arthritis

Objective Th17 has been shown to have a pivotal role in the development of arthritis. However, the role of IL-17 in the T cell-independent effector phase has not fully been examined. We investigated whether IL-17 is involved in the effector phase of arthritis by using K/BxN serum-induced arthritis model. Methods K/BxN serum was transferred into IL-17 knockout (KO) mice, SCID mice and their control mice, and arthritis was evaluated over time. In order to clarify the source of IL-17 in the effector phase, neutrophils or CD4+ T cells collected from IL-17 KO or control mice were injected into IL-17 KO recipient mice together with K/BxN serum. To examine if neutrophils secrete IL-17 upon stimulation, neutrophils were stimulated with immune complex in vitro and IL-17 in the supernatant was measured by ELISA. Results K/BxN serum-induced arthritis was much less severe in IL-17 KO mice than in WT mice. Since K/BxN serum-transferred SCID mice developed severe arthritis with high serum IL-17 concentration, we speculated neutrophils are the responsible player as an IL-17 source. When wild type (WT) but not IL-17 KO neutrophils were co-injected with K/BxN serum into IL-17 KO mice, arthritis was exacerbated, whereas co-injection of WT CD4+ T cells had no effect. In vitro, stimulation of neutrophils with immune complexcaused IL-17 secretion. Conclusions Neutrophils are essential as a source of IL-17 in the effector phase of arthritis. The trigger of secreting IL-17 from neutrophils may be immune complex.

PLD4 as a novel susceptibility gene for systemic sclerosis in a Japanese population

OBJECTIVE Systemic sclerosis (SSc) is an autoimmune disease for which multiple susceptibility genes have been reported. Genome-wide association studies have shown that large numbers of susceptibility genes are shared among autoimmune diseases. Recently, our group identified 9 novel susceptibility genes associated with rheumatoid arthritis (RA) in a Japanese population. The aim of this study was to elucidate whether the 18 genes that displayed associations or suggestive associations for RA in our previous study are associated with SSc in Japanese. METHODS We performed an association study that included 415 patients with SSc and 16,891 control subjects, followed by a replication study that included 315 patients and 21,054 control subjects. The 18 markers reported to display association with RA were analyzed for their associations with SSc in the first study, and 5 markers were further analyzed in the replication study. The inverse variance method was used to evaluate the associations of these markers with SSc in a combined study. RESULTS In the phospholipase D4 gene (PLD4), rs2841277 displayed a significant association with SSc in Japanese patients (P = 0.00017). We observed that rs2841280 in exon 2 of PLD4 was in strong linkage disequilibrium with rs2841277 and introduced an amino acid alteration. We also observed associations between SSc and rs6932056 in TNFAIP3 and rs2280381 in IRF8 (P = 0.0000095 and P = 0.0030, respectively), both of which displayed associations with SSc in a European population. CONCLUSION We determined that PLD4 is a novel susceptibility gene for SSc in Japanese, thus confirming the involvement of PLD4 in autoimmunity. Associations between SSc and TNFAIP3 or IRF8 were also detected in our Japanese population. SSc and RA appear to share relatively large proportions of their genetic backgrounds.