Atsuo Taniguchi

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.

Polymorphisms in the methylenetetrahydrofolate reductase gene were associated with both the efficacy and the toxicity of methotrexate used for the treatment of rheumatoid arthritis, as evidenced by single locus and haplotype analyses.

5,10-Methylenetetrahydrofolate reductase (MTHFR), a key enzyme involved in folate metabolism, has two common polymorphisms that affect enzyme activity. The objective of this study was to examine whether there was a correlation between the genotype or haplotype of the MTHFR gene and the efficacy or toxicity of methotrexate (MTX) in the treatment of rheumatoid arthritis. MTX-treated rheumatoid arthritis patients (n = 106) were selected from outpatient clinics and used for a retrospective study to examine the correlation between genotypes or haplotypes concerning polymorphisms of the MTHFR gene, and the efficacy or toxicity of MTX. Estimation of the haplotype frequencies was performed by maximum likelihood estimation based on expectation maximization algorithm. Single locus analysis examining each locus separately showed that patients with 1298C were receiving significantly lower doses of MTX compared to patients without [P < 0.05, relative risk (RR) = 2.18, 95% confidence interval (CI) 1.17-4.06], while a higher rate of overall MTX toxicity was observed in patients with 677T than those without (P < 0.05, RR = 1.25, 95% CI 1.05-1.49). An estimation of haplotype frequencies showed that there was no 677T-1298C haplotype in the population. Posterior distribution of the diplotype configuration for each individual was concentrated on a single configuration. Patients with the 677C-1298C haplotype were receiving lower doses of MTX than those without (P < 0.05, RR = 2.14, 95% CI 1.13-4.07), while subjects with 677T-1298A had a higher frequency of side-effects from MTX (P < 0.05, RR = 1.42, 95% CI 1.11-1.82). Both single locus and haplotype analyses suggest that polymorphisms within the MTHFR gene are associated with both the efficacy and toxicity of MTX in rheumatoid arthritis patients. Pharmacokinetic studies are necessary to prove the association.

Serum matrix metalloproteinase 3 as a predictor of the degree of joint destruction during the six months after measurement, in patients with early rheumatoid arthritis

OBJECTIVE To evaluate whether matrix metalloproteinase 3 (MMP-3), a proteinase that is expressed in rheumatoid synovial tissue and shows potent activity in degrading the proteoglycan of cartilage, plays a pivotal role in the joint destruction seen in early rheumatoid arthritis (RA). METHODS In a prospective study of patients with early RA, the relationship between the serum concentration of MMP-3, as determined by a sandwich enzyme immunoassay system, and the progression of joint destruction in patients with early RA, as measured by the Larsen radiologic score, was investigated. RESULTS Serum MMP-3 levels were elevated in the RA patients compared with healthy controls, not only in the late stage, but also in the early stage of the disease in patients whose duration of RA was <4 months. The serum MMP-3 level at entry into the study had a strong correlation with the Larsen score at 6 months and 12 months after entry (r = 0.58 and r = 0.49, respectively). Similarly, the serum MMP-3 level at 12 months and 24 months after entry showed a positive association with the Larsen score in the subsequent 6-12 months. Suppression of the serum MMP-3 level in the first year led to a decline in joint damage in the second year. CONCLUSION The serum concentration of MMP-3 is a useful marker for predicting bone damage in the early stage of RA, and the suppression of MMP-3 production may be an effective therapeutic approach for patients with early RA.

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.

Mortality and cause of death in Japanese patients with rheumatoid arthritis based on a large observational cohort, IORRA

Objectives: To investigate mortality, cause of death, and risk factors related to mortality in Japanese patients with rheumatoid arthritis (RA). Methods: The IORRA cohort is a large observational cohort established in 2000 at the Institute of Rheumatology, Tokyo Womens Medical University. Essentially, all RA patients were registered and clinical parameters were assessed biannually. For patients who failed to participate in subsequent surveys, simple queries were mailed to confirm survival. Standardized mortality ratios (SMRs) were calculated and mortality risk factors were analysed using a Cox proportional hazard model. Results: We analysed 7926 patients (81.9% females; mean age 56.3 ± 13.1 years; mean disease duration 8.5 ± 8.3 years) with RA who enrolled in IORRA from October 2000 to April 2007. During the observational period (35 443.0 person-years), 289 deaths were reported. Major causes of death included malignancies (24.2%), respiratory involvement (24.2%) including pneumonia (12.1%) and interstitial lung disease (ILD) (11.1%), cerebrovascular disease (8.0%), and myocardial infarction (7.6%). As death was not confirmed in all patients, the SMR was deduced to be between 1.46 [95% confidence interval (CI) 1.32–1.60] and 1.90 (95% CI 1.75–2.07) for all patients, between 1.45 (95% CI 1.22–1.70) and 1.70 (95% CI 1.45–1.97) for men, and between 1.46 (95% CI, 1.29–1.65) and 2.02 (95% CI 1.82–2.24) for women. Factors associated with increased mortality included male gender, older age, worse physical disability, positive rheumatoid factor (RF), corticosteroid use, and presence of ILD. Conclusion: The mortality of Japanese RA patients is comparable to that in previous reports from western countries, even though the causes of death were significantly different.

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Validation of the associations between single nucleotide polymorphisms or haplotypes and responses to disease-modifying antirheumatic drugs in patients with rheumatoid arthritis : a proposal for prospective pharmacogenomic study in clinical practice

Background For prevention of joint destruction in rheumatoid arthritis, optimal management of therapy with disease-modifying antirheumatic drugs is essential. Pharmacogenomic evidence, if reliable, may be incorporated in the treatment of rheumatoid arthritis to achieve a more efficient activity control with minimized adverse events. Methods We conducted retrospective studies to validate our previous three different results about the association between adverse events or efficacy of two different disease-modifying antirheumatic drugs and genomic variations. Association between single nucleotide polymorphisms in N-acetyltransferase 2 gene (NAT2) and adverse events by sulfasalazine and association between C677T or A1298C in 5,10-methylenetetrahydrofolate reductase gene (MTHFR) and responses to methotrexate were examined. Results Patients without the wild-type haplotype at NAT2 were more likely to suffer from overall adverse events [n=186, P=0.001, relative risk (RR) 3.31, 95% confidence interval (CI) 1.76–6.22] and severe adverse events (P=0.015, RR 24.6, 95% CI 2.37–254.53) by sulfasalazine. Patients with the T allele at C677T in MTHFR were more susceptible to overall adverse events (n=156, P=0.003; RR 2.4, 95% CI 1.29–4.55) while patients with the C allele at A1298C were less likely to be treated with a higher dose (>6 mg/week) of methotrexate in one year of treatment (n=159, P=0.008, RR 1.84, 95% CI 1.12–3.01). In all three association studies, the results were essentially the same as previously reported. Conclusion As three studies on the associations between genomic variations and adverse events or efficacy of two different disease-modifying antirheumatic drugs were replicated, the usefulness of the tests is worth being tested in clinical practice.

Sodium-dependent phosphate cotransporter type 1 sequence polymorphisms in male patients with gout

Objectives Molecular biological approaches have recently identified urate transporters in renal proximal tubular cells. Human sodium-dependent phosphate cotransporter type 1 encoded by SLC17A1 is a urate transporter localised to the renal proximal tubular cells and candidate molecule to secret urate from renal tubular cells to urine. This study investigated the roles of SLC17A1 in the development of gout. Patients and Methods Single nucleotide polymorphisms in the human SLC17A1 gene (rs1165176, rs1165151, rs1165153, rs1165196, rs1165209, rs1165215, rs1179086, rs3799344 and rs3757131) were selected, and an association study was conducted using male patients with gout (n=175) and male controls (n=595). Results There were significant differences between gout and control groups in the distribution of genotypes at rs1165196 (T806C; Ile269Thr, odds ratio (OR) 0.55, p=0.0035), rs1179086 (OR 0.57, p=0.0018) and rs3757131 (OR 0.54, p=0.0026). In controls, T806C alone had no effect on serum uric acid (sUA) levels. However, T806C showed significant interaction with a reduction of sUA in obese individuals (body mass index ≥25) using multiple regression analysis. Conclusions Our data suggest that SLC17A1 polymorphisms are associated with the development of gout.

Decrease in orthopaedic operations, including total joint replacements, in patients with rheumatoid arthritis between 2001 and 2007: data from Japanese outpatients in a single institute-based large observational cohort (IORRA)

Several studies from different countries show that the rate of orthopaedic surgery has decreased for patients with rheumatoid arthritis (RA) in recent years. In Sweden, there was a decrease in RA-related lower limb surgical procedures between 1987 and 2001,1 and in RA-related upper limb surgery between 1998 and 2004.2 Denmark has reported a decrease in the incidence of total hip arthroplasties due to RA,3 and the number of total joint replacement (TJR) operations and synovectomies decreased in the Norwegian population from 1994 to 2004.4 Japan has also reported the declining use of synovectomy surgery for patients with RA.5 These changes may reflect trends in disease severity, management and health outcomes in each country. Meanwhile, Sokka et al reported that the rate of TJR …

Control of renal uric acid excretion and gout.

Purpose of reviewImpaired renal uric acid excretion is the major mechanism of hyperuricemia in patients with primary gout. This review highlights recent advances in the knowledge of normal mechanisms of renal uric acid handling and derangement of these mechanisms in uric acid underexcretion. Recent findingsThe discovery of URAT1 has facilitated identification of other molecules potentially involved in uric acid transport in the renal tubules. Some of these molecules show gender differential expression in animal experiments. Sodium-dependent monocarboxylate cotransporters have been shown to transport lactate and butyrate, and may have roles in hyperuricemia associated with diabetic ketoacidosis and alcohol ingestion. Certain polymorphisms in SLC22A12 may be associated with the development of hyperuricemia or gout, although confirmation is needed. Mechanisms of hyperuricemia associated with uric acid underexcretion in patients with familial juvenile hyperuricemic nephropathy also remain to be clarified. Distal tubular salt wasting and compensatory upregulation of the resorption of sodium and uric acid in the proximal tubule may explain the hyperuricemia associated with this disorder. SummaryMuch progress has been made in understanding the mechanisms of renal uric acid handling. Elucidation of the mechanisms of hyperuricemia in patients with familial juvenile hyperuricemic nephropathy will shed light on the function of uromodulin, functional impairment of which eventually results in diminished uric acid excretion.