Erik J. M. Toonen

Tumour necrosis factor alpha -308G->A polymorphism is not associated with response to TNFalpha blockers in Caucasian patients with rheumatoid arthritis: systematic review and meta-analysis.

Background There is a need for biomarkers that can predict anti-tumour necrosis factor (anti-TNF) treatment outcome in patients with rheumatoid arthritis (RA). Several studies have suggested that the rare A allele of the tumour necrosis factor α (TNFA) −308G→A polymorphism could be associated with a poorer response to anti-TNF therapy. Nevertheless, these results remain controversial. Objective To determine by a meta-analysis whether the TNFA −308G→A polymorphism is associated with response to anti-TNF treatment in patients with RA. Methods A bibliographic search identified studies in which the TNFA −308G→A gene polymorphism was investigated in Caucasian patients with RA treated with anti-TNF agents. Complementary data were requested when the 28-joint count Disease Activity Score (DAS28) was not used as the primary outcome measure. Odds ratios (ORs) for response based on DAS28 and standardised mean difference (SMD) for mean improvement of DAS28 were calculated to assess the potential association between TNFA −308 genotypes and response to anti-TNF agents. Results The bibliographic search yielded 12 studies that met the inclusion criteria, which were supplemented with the data from a large Dutch cohort (n=426). The OR based on the 12 studies including 1721 patients was 1.24 (95% CI 0.98 to 1.56) and the SMD based on 11 studies including 2579 patients was −0.18 (95% CI −0.36 to 0.1). Subgroup analysis based on the two classes of anti-TNF agents did not demonstrate any association between TNFA −308 genotypes and anti-TNF treatment outcome. Conclusion According to this meta-analysis, the TNFA −308 polymorphism is not a predictor of the clinical response to anti-TNF treatment in RA.

Genome-wide association analysis of anti-TNF drug response in patients with rheumatoid arthritis

Background Treatment strategies blocking tumour necrosis factor (anti-TNF) have proven very successful in patients with rheumatoid arthritis (RA). However, a significant subset of patients does not respond for unknown reasons. Currently, there are no means of identifying these patients before treatment. This study was aimed at identifying genetic factors predicting anti-TNF treatment outcome in patients with RA using a genome-wide association approach. Methods We conducted a multistage, genome-wide association study with a primary analysis of 2 557 253 single-nucleotide polymorphisms (SNPs) in 882 patients with RA receiving anti-TNF therapy included through the Dutch Rheumatoid Arthritis Monitoring (DREAM) registry and the database of Apotheekzorg. Linear regression analysis of changes in the Disease Activity Score in 28 joints after 14 weeks of treatment was performed using an additive model. Markers with p<10−3 were selected for replication in 1821 patients from three independent cohorts. Pathway analysis including all SNPs with p<10−3 was performed using Ingenuity. Results 772 markers showed evidence of association with treatment outcome in the initial stage. Eight genetic loci showed improved p value in the overall meta-analysis compared with the first stage, three of which (rs1568885, rs1813443 and rs4411591) showed directional consistency over all four cohorts studied. We were unable to replicate markers previously reported to be associated with anti-TNF outcome. Network analysis indicated strong involvement of biological processes underlying inflammatory response and cell morphology. Conclusions Using a multistage strategy, we have identified eight genetic loci associated with response to anti-TNF treatment. Further studies are required to validate these findings in additional patient collections.

Pharmacogenetics of anti-TNF treatment in patients with rheumatoid arthritis

TNF-blocking strategies are widely used in the treatment of rheumatoid arthritis (RA). Three anti-TNF agents are registered for use in RA: etanercept, infliximab and adalimumab. Although anti-TNF therapy is very effective in controlling disease activity and slowing down radiological damage, prolonged response is only seen in approximately 70% of the patients. The causes for nonresponse in the remaining patients have not yet been elucidated. Pharmacogenetic studies focusing on genes involved in RA etiology (and/or progression) and in the pharmacokinetics of TNF-blocking agents have identified markers associated with anti-TNF treatment outcome. In the future, more exhaustive, less hypothesis-driven search strategies are expected to discover additional markers. Identification of these markers might be viewed as the first step towards tailored TNF-blocking therapy for patients with RA. Nevertheless, replication and large prospective studies will be needed to demonstrate the validity of the identified genetic markers before implementation into daily clinical practice.

Validation Study of Existing Gene Expression Signatures for Anti-TNF Treatment in Patients with Rheumatoid Arthritis

So far, there are no means of identifying rheumatoid arthritis (RA) patients who will fail to respond to tumour necrosis factor blocking agents (anti-TNF), prior to treatment. We set out to validate eight previously reported gene expression signatures predicting therapy outcome. Genome-wide expression profiling using Affymetrix GeneChip Exon 1.0 ST arrays was performed on RNA isolated from whole blood of 42 RA patients starting treatment with infliximab or adalimumab. Clinical response according to EULAR criteria was determined at week 14 of therapy. Genes that have been reported to be associated with anti-TNF treatment were extracted from our dataset. K-means partition clustering was performed to assess the predictive value of the gene-sets. We performed a hypothesis-driven analysis of the dataset using eight existing gene sets predictive of anti-TNF treatment outcome. The set that performed best reached a sensitivity of 71% and a specificity of 61%, for classifying the patients in the current study. We successfully validated one of eight previously reported predictive expression profile. This replicated expression signature is a good starting point for developing a prediction model for anti-TNF treatment outcome that can be used in a daily clinical setting. Our results confirm that gene expression profiling prior to treatment is a useful tool to predict anti-TNF (non) response.

The tumour necrosis factor receptor superfamily member 1b 676T>G polymorphism in relation to response to infliximab and adalimumab treatment and disease severity in rheumatoid arthritis

Objective: To assess the effect of a functional polymorphism (676T>G, M196R) in the tumour necrosis factor receptor super family 1b (TNFSF1b) gene on disease activity, radiological joint damage and response to infliximab and adalimumab treatment in patients with rheumatoid arthritis (RA). Methods: Two cohorts of patients with RA were genotyped for the 676T>G polymorphism (rs1061622) in exon 6 of the TNFSF1b gene by restriction fragment length polymorphism analysis. One cohort (n = 234) included patients from the Dutch Rheumatoid Arthritis Monitoring register with detailed information on their response to anti-TNF therapy (infliximab and adalimumab), the other cohort comprised patients from a long-term observational early inception cohort at our centre (n = 248). Results: The 676T>G polymorphism was not associated with anti-TNF response after 3 or 6 months of treatment. Linear regression analysis showed no significant difference in the progression of radiological joint damage during the first 3 and 6 years of disease between the three genotype groups (TT, TG and GG). Additionally, no difference in mean disease activity between genotypes was seen after 3 and 6 years of disease. Conclusion: Despite its demonstrated functionality, the 676T>G polymorphism in the TNFSF1b gene does not have a major role in either the response to anti-TNF therapy or in the disease severity or radiological progression in RA.

HMOX1 promoter polymorphism modulates the relationship between disease activity and joint damage in rheumatoid arthritis.

OBJECTIVE The guanine-thymidine (GT)n repeat in the HMOX1 promoter determines the level of induction of the heme-degrading enzyme heme oxygenase 1 (HO-1), which protects against inflammatory and oxidative stress. In individuals with short (GT)n repeats (where n < 25; SS genotype), higher levels of HO-1 activity are induced more rapidly than in those with long (GT)n repeats (where n > or = 25; LL genotype). Recently, it was demonstrated that HO-1 activity protects against the onset of rheumatoid arthritis (RA). The aim of this study was to determine whether the (GT)n-repeat length within the HMOX1 promoter region is associated with RA disease severity and radiographic joint damage. METHODS A cohort of 325 well-characterized RA patients and 273 controls was investigated by DNA fragment-length analysis for the association of (GT)n repeats in the HMOX1 promoter region with RA disease susceptibility and severity. RESULTS Although no significant differences in genotype or allele frequency were found between controls and RA patients, the odds ratios corresponded well to those in the previously described cohort. Among patients, those carrying the SS genotype had a more favorable radiographic outcome over 9 years than those carrying the LL genotype. This was unexpected since no differences in disease activity were found between the genotypes or alleles. CONCLUSION Patients with the SS genotype have a better long-term radiographic outcome despite poor prognostic markers at baseline and despite disease activity at followup similar to that of patients with the LL genotype. This suggests that the HMOX1/HO-1 system is involved in the uncoupling of disease activity and joint damage and may provide a novel target for the treatment of RA.

Gene expression profiling in rheumatoid arthritis: current concepts and future directions

Over the last years microarray technologies have generated new perspectives for the high-throughput analysis of biological systems. Nowadays, it is possible to monitor thousands of genes in a single experiment. This molecular profiling technology combined with standardised and validated clinical measurements can allow a more precise characterisation of a patient’s phenotype, and may lead to the design of therapeutic protocols and procedures better tailored to an individual patient’s needs. In this report we provide an overview of expression profiling studies in rheumatoid arthritis (RA). RA is a chronic inflammatory disease in which both genetic and environmental factors are involved. The precise molecular mechanisms underlying RA are not fully understood. A systematic literature search revealed nine array-based expression profiling studies in patients with RA. Findings from these studies were compared with those of linkage and genome-wide association (GWA) studies. Although we observed many differences in study design, analysis and interpretation of results between the different studies, we extracted two sets of genes: (1) those differentially expressed in more than one study, and (2) genes differentially expressed in at least one of the reviewed studies and present in RA linkage or GWA loci. We suggest that both sets of genes include interesting candidate genes for further study in RA.

Genetic variants in toll-like receptors are not associated with rheumatoid arthritis susceptibility or anti-tumour necrosis factor treatment outcome.

Background Several studies point to a role of Toll-like receptors (TLRs) in the development of rheumatoid arthritis (RA). We investigated if genetic variants in TLR genes are associated with RA and response to tumour necrosis factor blocking (anti-TNF) medication. Methodology and Principal Findings 22 single nucleotide polymorphisms (SNPs) in seven TLR genes were genotyped in a Dutch cohort consisting of 378 RA patients and 294 controls. Significantly associated variants were investigated in replication cohorts from The Netherlands, United Kingdom and Sweden (2877 RA patients and 2025 controls). 182 of the Dutch patients were treated with anti-TNF medication. Using these patients and a replication cohort (269 Swedish patients) we analysed if genetic variants in TLR genes were associated with anti-TNF outcome. In the discovery phase of the study we found a significant association of SNPs rs2072493 in TLR5 and rs3853839 in TLR7 with RA disease susceptibility. Meta-analysis of discovery and replication cohorts did not confirm these findings. SNP rs2072493 in TLR5 was associated with anti-TNF outcome in the Dutch but not in the Swedish population. Conclusion We conclude that genetic variants in TLRs do not play a major role in susceptibility for developing RA nor in anti-TNF treatment outcome in a Caucasian population.

Meta-analysis identified the TNFA -308G > A promoter polymorphism as a risk factor for disease severity in patients with rheumatoid arthritis.

IntroductionThe goal of this study is to investigate whether the -308G > A promoter polymorphism in the tumor necrosis factor alpha (TNFA) gene is associated with disease severity and radiologic joint damage in a large cohort of patients with rheumatoid arthritis (RA).MethodsA long-term observational early RA inception cohort (n = 208) with detailed information about disease activity and radiologic damage after 3, 6 and 9 years of disease was genotyped for the TNFA -308G > A promoter polymorphism (rs1800629). A longitudinal regression analysis was performed to assess the effect of genotype on RA disease severity and joint damage. Subsequently, a meta-analysis, including all publically available data, was performed to further test the association between joint erosions and the TNFA polymorphism. To learn more about the mechanism behind the effect of the polymorphism, RNA isolated from peripheral blood from RA patients (n = 66) was used for TNFA gene expression analysis by quantitative PCR.ResultsLongitudinal regression analysis with correction for gender and disease activity showed a significant difference in total joint damage between GG and GA+AA genotype groups (P = 0.002), which was stable over time. The meta-analysis, which included 2,053 patients, confirmed an association of the genetic variant with the development of erosions (odds ratio 0.78, 95% CI 0.62, 0.98). No significant differences in TNFA gene expression were observed for the different genotypes, confirming earlier findings in healthy individuals.ConclusionsOur data confirm that the TNFA -308G > A promoter polymorphism is associated with joint damage in patients with RA. This is not mediated by differences in TNFA gene expression between genotypes.

Multiplex Screening of 22 Single-Nucleotide Polymorphisms in 7 Toll-like Receptors: An Association Study in Rheumatoid Arthritis

Objective. Toll-like receptors (TLR) have been implicated in the pathogenesis of arthritis. We investigated the role of functional variants of TLR in the disease phenotype and severity of rheumatoid arthritis (RA). Methods. All patients from a longterm observational inception cohort (n = 319) were genotyped for 22 single-nucleotide polymorphisms (SNP) in TLR2, 3, 4, 5, 7, 8, and 9 using multiplex assays. Clinical characteristics including sex, age at disease onset, rheumatoid factor (RF), and shared epitope positivity and disease activity score and radiological progression were taken into account. Genotypes were analyzed for association with Disease Activity Scores (DAS28) and joint damage (Rau scores) at 3 and 6 years. Results. After Bonferroni correction, there was a moderate association between RF positivity and TLR8-rs5741883. No other TLR variant was significantly associated with any RA clinical characteristics. Conclusion. Using a large inception cohort and strict statistical evaluation, we could not identify an association between functional TLR variants and RA phenotype and disease severity. This suggests the functional TLR variants do not play a major role in RA phenotype and disease severity.