Investigating CD11c expression as a potential genomic biomarker of response to TNF inhibitor biologics in whole blood rheumatoid arthritis samples.

INTRODUCTION: Gene expression profiling is rapidly becoming a useful and informative tool in a much needed area of research. Identifying patients as to whether they will respond or not to a given treatment before prescription is not only essential to optimise treatment outcome but also to lessen the economic burden that such drugs can have on healthcare resources. In rheumatoid arthritis (RA), there is of yet no genetic/genomic biomarker which can accurately predict response to TNF inhibitor biologics prior to treatment, despite much interest in this area. Multiple studies have reported findings on potential candidate genes; however, due to relatively small sample sizes or lack of sufficient validation, results have been disappointingly inconsistent. The aim of this research was to further explore the predictive value of a previously reported association between CD11c expression and response to the TNF inhibitor biologics, adalimumab and etanercept. METHODS: Real-time qPCR was performed using whole blood RNA samples obtained from seventy-five rheumatoid arthritis patients about to commence treatment with a TNF inhibitor biologic drug, whose response status was determined at 3-month follow-up using the EULAR classification criteria. Relative quantification of CD11c using the comparative CT method outputted differential expression between good-responders and non-responders as a fold-change. RESULTS: Relative expression of CD11c in patients receiving TNF inhibitor biologics yielded a decrease of 1.025 fold in good-responders as compared to non-responders (p-value = 0.36). Upon stratification of patients dependent upon the specific drug administered, adalimumab or etanercept, similar findings to the full cohort were observed, decreases of 1.015 (p-value = 0.33) and 1.032 fold (p-value = 0.13) in good-responders compared to non-responders, respectively. CONCLUSION: The results from this study reveal that CD11c expression does not correlate with response to TNF inhibitor biologics when tested for within pre-treatment whole blood samples of rheumatoid arthritis patients.

Genome-wide association meta-analyses to identify common genetic variants associated with hallux valgus in Caucasian and African Americans.

OBJECTIVE: Hallux valgus (HV) affects ∼36% of Caucasian adults. Although considered highly heritable, the underlying genetic determinants are unclear. We conducted the first genome-wide association study (GWAS) aimed to identify genetic variants associated with HV. METHODS: HV was assessed in three Caucasian cohorts (n=2263, n=915 and n=1231 participants, respectively). In each cohort, a GWAS was conducted using 2.5 M imputed SNPs. Mixed-effect regression with the additive genetic model adjusted for age, sex, weight and within-family correlations was used for both sex-specific and combined analyses. To combine GWAS results across cohorts, fixed-effect inverse-variance meta-analyses were used. Following meta-analyses, top-associated findings were also examined in an African American cohort (n=327). RESULTS: The proportion of HV variance explained by genome-wide genotyped SNPs was 50% in men and 48% in women. A higher proportion of genetic determinants of HV were sex specific. The most significantly associated SNP in men was rs9675316 located on chr17q23-a24 near the AXIN2 gene (p=0.000000546×10(-7)); the most significantly associated SNP in women was rs7996797 located on chr13q14.1-q14.2 near the ESD gene (p=0.000000721×10(-7)). Genome-wide significant SNP-by-sex interaction was found for SNP rs1563374 located on chr11p15.1 near the MRGPRX3 gene (interaction p value =0.0000000041×10(-9)). The association signals diminished when combining men and women. CONCLUSIONS: The findings suggest that the potential pathophysiological mechanisms of HV are complex and strongly underlined by sex-specific interactions. The identified genetic variants imply contribution of biological pathways observed in osteoarthritis as well as new pathways, influencing skeletal development and inflammation.

Association of a complement receptor 1 gene variant with baseline erythrocyte sedimentation rate levels in patients starting anti-TNF therapy in a UK rheumatoid arthritis cohort: results from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate cohort.

Eligibility for anti-tumour necrosis factor (TNF) therapy in most European countries is restricted to severe, active rheumatoid arthritis (RA). The DAS28 score is a marker of disease severity and incorporates one of two inflammatory markers, erythrocyte sedimentation rate (ESR) or C-reactive protein. We aimed to determine the relation between genetic variants known to affect ESR and levels of ESR in patients with active RA. DNA samples were genotyped for four single-nucleotide polymorphisms (SNPs) rs7527798 (CR1L), rs6691117 (CR1), rs10903129 (TMEM57) and rs1043879 (C1orf63). The association between SNPs and baseline ESR, baseline DAS28-ESR, and change in DAS28-ESR was evaluated. Baseline ESR was significantly associated with CR1 rs6691117 genotype (P=0.01). No correlation was identified between baseline DAS28-ESR or change in DAS28-ESR. In conclusion, genetic variation in the gene encoding CR1 may alter ESR levels but not DAS28-ESR, indicating no adjustment for CR1 genotype is required in the assessment of patients with severe active RA.

Identification of BACH2 and RAD51B as rheumatoid arthritis susceptibility loci in a meta-analysis of genome-wide data.

OBJECTIVE: A recent high-density fine-mapping (ImmunoChip) study of genetic associations in rheumatoid arthritis (RA) identified 14 risk loci with validated genome-wide significance, as well as a number of loci showing associations suggestive of significance (P = 5 × 10(-5) < 5 × 10(-8)), but these have yet to be replicated. The aim of this study was to determine whether these potentially significant loci are involved in the pathogenesis of RA, and to explore whether any of the loci are associated with a specific RA serotype. METHODS: A total of 16 single-nucleotide polymorphisms (SNPs) were selected for genotyping and association analyses in 2 independent validation cohorts, comprising 6,106 RA cases and 4,290 controls. A meta-analysis of the data from the original ImmunoChip discovery cohort and from both validation cohorts was carried out, for a combined total of 17,581 RA cases and 20,160 controls. In addition, stratified analysis of patient subsets, defined according to their anti-cyclic citrullinated peptide (anti-CCP) antibody status, was performed. RESULTS: A significant association with RA risk (P < 0.05) was replicated for 6 of the SNPs assessed in the validation cohorts. All SNPs in the validation study had odds ratios (ORs) for RA susceptibility in the same direction as those in the ImmunoChip discovery study. One SNP, rs72928038, mapping to an intron of BACH2, achieved genome-wide significance in the meta-analysis (P = 1.2 × 10(-8), OR 1.12), and a second SNP, rs911263, mapping to an intron of RAD51B, was significantly associated in the anti-CCP-positive RA subgroup (P = 4 × 10(-8), OR 0.89), confirming that both are RA susceptibility loci. CONCLUSION: This study provides robust evidence for an association of RA susceptibility with genes involved in B cell differentiation (BACH2) and DNA repair (RAD51B). The finding that the RAD51B gene exhibited different associations based on serologic subtype adds to the expanding knowledge base in defining subgroups of RA.

Association of genetic variants in the IL4 and IL4R genes with the severity of joint damage in rheumatoid arthritis: a study in seven cohorts.

OBJECTIVE: The progression of joint destruction in rheumatoid arthritis (RA) is determined by genetic factors. Changes in IL4 and IL4R genes have been associated with RA severity, but this finding has not been replicated. This study was undertaken to investigate the association between IL4- and IL4R-tagging single-nucleotide polymorphisms (SNPs) and the progression rate of joint damage in RA in a multicohort candidate gene study. METHODS: IL4- and IL4R-tagging SNPs (n = 8 and 39, respectively) were genotyped in 600 RA patients for whom 2,846 sets of radiographs of the hands and feet were obtained during 7 years of followup. Subsequently, SNPs significantly associated with the progression of joint damage were genotyped and studied in relation to 3,415 radiographs of 1,953 RA patients; these included data sets from Groningen (The Netherlands), Lund (Sweden), Sheffield (UK), the North American Rheumatoid Arthritis Consortium (US), Wichita (US), and the National Data Bank (US). The relative increase in progression rate per year in the presence of a genotype was determined in each cohort. An inverse variance weighting meta-analysis was performed on the 6 data sets that together formed the replication phase. RESULTS: In the discovery phase, none of the IL4 SNPs and 7 of the IL4R SNPs were significantly associated with the joint damage progression rate. In the replication phase, 2 SNPs in the IL4R gene were significantly associated with the joint damage progression rate (rs1805011 [P = 0.02] and rs1119132 [P = 0.001]). CONCLUSION: Genetic variants in IL4R were identified, and their association with the progression rate of joint damage in RA was independently replicated.

A genetic variant in granzyme B is associated with progression of joint destruction in rheumatoid arthritis.

OBJECTIVE: Genetic factors account for an estimated 45-58% of the variance in joint destruction in rheumatoid arthritis (RA). The serine proteinase granzyme B induces target cell apoptosis, and several in vitro studies suggest that granzyme B is involved in apoptosis of chondrocytes. Serum levels of granzyme B are increased in RA and are also associated with radiographic erosions. The aim of this study was to investigate GZMB as a candidate gene accounting for the severity of joint destruction in RA. METHODS: A total of 1,418 patients with 4,885 radiograph sets of the hands and feet from 4 independent cohorts were studied. First, explorative analyses were performed in 600 RA patients in the Leiden Early Arthritis Clinic cohort. Fifteen single-nucleotide polymorphisms (SNPs) tagging GZMB were tested. Significantly associated SNPs were genotyped in data sets representing patients from the Groningen, Sheffield, and Lund cohorts. In each data set, the relative increase in the annual rate of progression in the presence of a genotype was assessed. Data were summarized in a meta-analysis. The association of GZMB with the RNA expression level of the GZMB genomic region was tested by mapping expression quantitative trait loci (QTLs) on 1,469 whole blood samples. RESULTS: SNP rs8192916 was significantly associated with the rate of joint destruction in the first cohort and in the meta-analysis of all data sets. Patients homozygous for the minor allele of rs8192916 had a higher rate of joint destruction per year compared with other patients (P = 7.8 × 10(-4)). Expression QTL of GZMB identified higher expression in the presence of the minor allele of rs8192916 (P = 2.27 × 10(-5)). CONCLUSION: SNP rs8192916 located in GZMB is associated with the progression of joint destruction in RA as well as with RNA expression in whole blood.

Genetic variants in IL15 associate with progression of joint destruction in rheumatoid arthritis: a multicohort study.

BACKGROUND: Interleukin (IL)-15 levels are increased in serum, synovium and bone marrow of patients with rheumatoid arthritis (RA). IL-15 influences both the innate and the adaptive immune response; its major role is activation and proliferation of T cells. There are also emerging data that IL-15 affects osteoclastogenesis. The authors investigated the association of genetic variants in IL15 with the rate of joint destruction in RA. METHOD: 1418 patients with 4885 x-ray sets of both hands and feet of four independent data sets were studied. First, explorative analyses were performed on 600 patients with early RA enrolled in the Leiden Early Arthritis Clinic. Twenty-five single-nucleotide polymorphisms (SNPs) tagging IL-15 were tested. Second, SNPs with significant associations in the explorative phase were genotyped in data sets from Groningen, Sheffield and Lund. In each data set, the relative increase of the progression rate per year in the presence of a genotype was assessed. Subsequently, data were summarised in an inverse weighting meta-analysis. RESULTS: Five SNPs were significantly associated with rate of joint destruction in phase 1 and typed in the other data sets. Patients homozygous for rs7667746, rs7665842, rs2322182, rs6821171 and rs4371699 had respectively 0.94-, 1.04-, 1.09-, 1.09- and 1.09-fold rate of joint destruction compared to other patients (p=4.0×10(-6), p=3.8×10(-4), p=5.0×10(-3), p=5.0×10(-3) and p=9.4×10(-3)). DISCUSSION: Independent replication was not obtained, possibly due to insufficient power. Meta-analyses of all data sets combined resulted in significant results for four SNPs (rs7667746, p<0.001; rs7665842, p<0.001; rs4371699, p=0.01; rs6821171, p=0.01). These SNPs were also significant after correction for multiple testing. CONCLUSION: Genetic variants in IL-15 are associated with progression of joint destruction in RA.

A spectrum of susceptibility to rheumatoid arthritis within HLA-DRB1: stratification by autoantibody status in a large UK population.

Previously-proposed rheumatoid arthritis (RA) HLA-DRB1 susceptibility and protective models were compared, based on amino acids at positions 67-74 and autoantibody combinations. 3 657 RA patients and 1 357 controls were studied using logistic regression, with secondary stratification by anti-citrullinated peptide antibodies(ACPA) and rheumatoid factor(RF). Susceptibility models were based on previously defined HLA-DRB1 shared epitope(SE) subgroups. (70)DERAA(74), D(70) and I(67) protective models were compared, adjusting for HLA-DRB1 SE. A hierarchy of risk was observed within the HLA-DRB1 SE, particularly for ACPA-positive and RF-positive RA: HLA-DRB1(*)0401∼(*)0404>(*)0101∼(*)1001 ((*)0404>(*)0101: P=0.0003). HLA-DRB1(*)0401/(*)0404 compound heterozygosity conferred a risk similar to (*)0401 homozygosity (P=0.70). Protective effects of D(70) and I(67) were similar. Predictions of the D(70) model fitted the data better than those of the I(67) model. The protective effect of D(70) showed a gene-dose effect (OR 0.82, 95% CI 0.73-0.92, P=5.8 × 10(-4)), but was only seen in RA patients positive for RF or ACPA. HLA-DRB1 SE alleles were also associated with ACPA-negative, RF-positive RA (OR 1.42 (1.15-1.76), P=0.0012). In conclusion, HLA-DRB1 SE alleles show heterogeneity in RA susceptibility; their major effect appears to be mediated by ACPA positivity, but a significant association of HLA-DRB1 SE with RF-positive, ACPA-negative RA was also observed. D(70) specifically protected against antibody-positive RA.

Genetic influences modulating the radiological severity of rheumatoid arthritis.

This review focuses on the contribution of genetic markers to the severity of radiological damage in rheumatoid arthritis (RA). Currently available biomarkers of more severe disease include elevated erythrocyte sedimentation rates or C-reactive protein levels and rheumatoid factor (RF) or anticyclic citrullinated protein antibodies positivity; however, these biomarkers explain a relatively modest proportion of the variance in radiological damage. An important role of genetic factors on RA severity has recently emerged but studies to date have generally been of low statistical power and many have not been replicated. Genetic markers have a number of advantages over conventional biomarkers; genotypes are stable, measurable at disease onset, remain unchanged by treatment and are amenable to high-throughput assays. The recent advances in genome-wide genetic analysis should lead to a more comprehensive understanding of RA severity genes. This knowledge could be used, along with existing biomarkers, to therapeutically target subjects at risk of poor radiological outcome.

Pharmacokinetic/pharmacodynamic modelling of 2-acetyl-4(5)-tetrahydroxybutyl imidazole-induced peripheral lymphocyte sequestration through increasing lymphoid sphingosine 1-phosphate.

2-Acetyl-4(5)-tetrahydroxybutyl imidazole (THI) has been shown to reduce rodent peripheral blood lymphocytes through increasing lymphoid sphingosine 1-phosphate (S1P) by inhibiting S1P lyase. The objective of this study was to characterize the relationship between systemic THI exposure, splenic S1P concentrations, and lymphopenia in rats. Following the oral administration of 10 and 100 mg kg(-1) THI to male rats, THI was rapidly absorbed and reached a plasma peak level at 1 h post-dosing. Splenic S1P increased and reached the peak level at 24 h. Blood lymphocyte count decreased as the splenic S1P level increased. THI plasma concentration was linked to splenic S1P concentration using an indirect model incorporated with a four-step signal transduction model. In turn, the S1P level was directly coupled with blood lymphocyte number. The integrated model simultaneously captured the splenic S1P and blood lymphocyte responses. This pharmacokinetic-biomarker-pharmacodynamic model resolved the remarkable discrepancy between plasma THI concentration and the pharmacological response and quantitatively described the relationship of THI exposure, S1P, and lymphopenic response.

A gain of function polymorphism in the interleukin 6 receptor influences RA susceptibility.

OBJECTIVES: To investigate the possible role of a functional polymorphism in the soluble interleukin 6 receptor (sIL-6R) gene in the genetic background of rheumatoid arthritis (RA). METHODS: An association between disease status and the sIL-6R rs8192284 (A358D) variant was tested in 965 patients with RA and 988 unrelated healthy controls. Odds ratios (ORs) for disease were calculated with asymptotic 95% CI; p values <0.05 were considered statistically significant after adjustment for multiple testing. To determine the relationship between protein levels and IL-6R A358D genotype, the protein levels of sIL-6R in 100 plasma samples from healthy controls were measured using an ELISA and compared across the genotype groups. RESULTS: The allele frequency of the C allele (alanine) was lower in cases than in controls (38.4% vs 41.7%, p=0.04, OR 0.9, 95% CI 0.8 to 1.0), as were the CC/AC genotypes compared with AA genotype frequencies (61.0% in RA cases vs 67.5% in controls, p=0.004, OR 0.8, 95% CI 0.6 to 0.9). Plasma levels of sIL-6R differed significantly according to genotype in the controls: 17.00 + or - 2.03 ng/ml for A/A, 20.08 + or - 1.83 ng/ml for A/C and 21.57 + or - 2.10 ng/ml for C/C (p=0.0001). CONCLUSION: These data suggest a role for genetically determined lower sIL-6R levels as a risk factor for RA. The proinflammatory role of the IL6 system in established RA has been highlighted by the use of anti-sIL-6R antibodies. However, the findings of this study suggest a protective effect of IL6 on the risk of developing RA.

Evidence of epistasis between interleukin 1 and selenoprotein-S with susceptibility to rheumatoid arthritis.

OBJECTIVE: Selenoprotein-S (SELS) is involved in the stress response within the endoplasmic reticulum (ER) and inflammation. Recently, promoter variants in the SELS gene were shown to be associated with plasma levels of interleukin (IL)6, IL1beta and tumour necrosis factor (TNF). It was hypothesised that these variants could influence rheumatoid arthritis (RA) susceptibility and may interact with functional single nucleotide polymorphisms (SNPs) in the genes for IL1, IL6 and TNF. METHODS: Genotyping was performed in 988 unrelated healthy controls and 965 patients with RA. Stratified analysis was used to test for interactions. Single gene effects and evidence of epistasis were investigated using the Mantel-Haenszel (M-H) test and the linkage disequilibrium (LD)-based statistic. RESULTS: No association of SELS -105 genotype and RA susceptibility was detected. Stratification of SELS -105 genotypes by IL1 -511 genotypes showed that the disease risk (comparing AA/GA to GG at the SELS -105 locus) in individuals with the GG/AG genotype at the IL1beta -511 locus was significantly lower than that in individuals having the AA genotype at the IL1beta -511 locus (odds ratio (OR): 0.9 and 2.3, respectively; p = 0.004 by M-H test). Significant epistasis was also detected using the LD-based statistic (p = <0.001). No interaction was observed between SELS -105 and IL6 or TNF variants. CONCLUSION: Our results reveal evidence of strong epistasis in two genes in the IL1 production pathway and highlight the potential importance of gene-gene interactions in the pathogenesis of RA.

Association of rheumatoid factor and anti-cyclic citrullinated peptide positivity, but not carriage of shared epitope or PTPN22 susceptibility variants, with anti-tumour necrosis factor response in rheumatoid arthritis.

OBJECTIVE: To determine whether rheumatoid factor (RF), anti-cyclic citrullinated peptide (CCP) antibodies, or carriage of shared epitope (SE) and PTPN22 genetic susceptibility variants predict response to therapy in patients with rheumatoid arthritis (RA) treated with anti-tumour necrosis factor (TNF) agents. METHODS: UK-wide multicentre collaborations were established to recruit a large cohort of patients treated with anti-TNF drugs for RA. Serum RF, anti-CCP antibody and SE status were determined using commercially available kits. PTPN22 R620W genotyping was performed by Sequenom MassArray. Linear regression analyses were performed to investigate the role of these four factors in predicting response to treatment by 6 months, defined as the absolute change in 28-joint Disease Activity Score (DAS28). RESULTS: Of the 642 patients analysed, 46% received infliximab, 43% etanercept and 11% adalimumab. In all, 89% and 82% of patients were RF and anti-CCP positive, respectively. Patients that were RF negative had a 0.48 (95% CI 0.08 to 0.87) greater mean improvement in DAS28 compared to patients that were RF positive. A better response was also seen among patients that were anti-CCP negative. No association was demonstrated between drug response and SE or PTPN22 620W carriage. CONCLUSION: The presence of RF or anti-CCP antibodies was associated with a reduced response to anti-TNF drugs. However, these antibodies only account for a small proportion of the variance in treatment response. It is likely that genetic factors will contribute to treatment response, but these do not include the well established RA susceptibility loci, SE and PTPN22.

Stem cell transplantation in rheumatoid arthritis.

The therapeutic potential of high dose cytotoxic therapy and stem cell transplantation (SCT) in severe rheumatoid arthritis (RA) was originally supported by animal studies and serendipitous clinical cases where allogeneic and autologous procedures were shown to ameliorate and potentially cure the disease. Phase I and Phase II clinical studies established the feasibility, safety and efficacy of autologous stem cell mobilisation and transplantation. Although it was clear that the effects of high dose chemotherapy and autologous SCT could safely achieve profound responses, sustained control of disease usually required the reintroduction of disease modifying agents. Responses were improved with dose escalation of the conditioning regimen, and also with post-SCT therapy, such as rituximab, but were not observed with graft manipulation. Phase III studies were attempted, but recruitment was compromised by the increasingly widespread use of biological anti-rheumatic agents. Autologous SCT is now only reasonably considered in relatively rare patients whose disease has resisted conventional and biological treatments, and small numbers of cases continue to be registered with the EBMT. Occasional patients treated with allogeneic and syngeneic SCT continue to stimulate academic interest, particularly as some appear to be cured, but significant logistical and toxicity issues mean that routine and widespread application is unrealistic. In summary, SCT continues to have a limited therapeutic potential in rare patients with RA refractory to modern therapy and sufficient fitness for the procedure. From a scientific perspective, ablation of the dysfunctional rheumatoid immune system and its reconstruction with SCT has provided useful insights into the pathophysiology of RA.

The effects of contemporary processes in maintaining the genetic structure of western song sparrows (Melospiza melodia).

Historic events and contemporary processes work in concert to create and maintain geographically partitioned variation and are instrumental in the generation of biodiversity. We sought to gain a better understanding of how contemporary processes such as movement and isolation influence the genetic structure of widely distributed vagile species such as birds. Song sparrows (Melospiza melodia) in western North America provide a natural system for examining the genetics of populations that have different patterns of geographic isolation and migratory behavior. We examined the population genetics of 576 song sparrows from 23 populations using seven microsatellite loci to assess genetic differentiation among populations and to estimate the effects of drift and immigration (gene flow) on each population. Sedentary, isolated populations were characterized by low levels of immigration and high levels of genetic drift, whereas those populations less isolated displayed signals of high gene flow and little differentiation from other populations. Contemporary dispersal rates from migratory populations, estimated by assignment test, were higher and occurred over larger distances than dispersal from sedentary populations but were also probably too low to counter the effects of drift in most populations. We suggest that geographic isolation and limited gene flow facilitated by migratory behavior are responsible for maintaining observed levels of differentiation among Pacific coastal song sparrow populations.

Absence of association of asporin polymorphisms and osteoarthritis susceptibility in US Caucasians.

OBJECTIVE: An association between osteoarthritis (OA) and functional polymorphisms in the aspartic acid (d) repeat of the asporin (ASPN) gene was reported in Japanese and Han Chinese populations. The aim of this study was to assess the association of variants in the ASPN gene with the presence of radiographic hand and/or knee OA in a US Caucasian population. METHODS: Ten single nucleotide polymorphisms (SNPs) within the ASPN gene were genotyped in 775 affected siblings with radiographically confirmed hand and/or knee OA, and the allelic, genotypic and haplotypic association results were examined. RESULTS: One variant (SNP RS7033979) showed nominal evidence of association with both hand OA (P=0.042) and knee OA (P=0.032). Four additional SNPs showed nominal evidence of association with knee OA only. These associations were only observed with genotypic tests; the corresponding allelic and haplotype tests did not corroborate the single-point association results. CONCLUSION: These data suggest that polymorphisms within ASPN are not a major influence in susceptibility to hand or knee OA in US Caucasians.

The Mal/TIRAP S180L and TLR4 G299D polymorphisms are not associated with susceptibility to, or severity of, rheumatoid arthritis.

BACKGROUND: Toll-like receptors (TLRs), including TLR4, have been implicated in the pathogenesis of rheumatoid arthritis (RA). Signalling by these receptors involves interactions with intracellular proteins, including the MyD88 adapter-like (Mal) protein. Recently, a polymorphism (Mal S180L) has been described which contributes to susceptibility to common infectious diseases and inhibits proinflammatory cytokine production. A non-synonymous variant in the extracellular domain of TLR4 (G299D) has been shown to interrupt TLR4-mediated signalling, resulting in endotoxin hyporesponsiveness. OBJECTIVE: To investigate the role of TLR4 G299D and Mal S180L variants in RA. METHODS: A total of 964 Caucasians with RA and 965 controls were genotyped. Deviation from Hardy-Weinberg equilibrium was tested for each single nucleotide polymorphism in cases and controls separately using a chi(2) test with a threshold of p<0.05. The odd ratios were calculated with asymptotic 95% confidence intervals, and p values <0.05 were considered significant. Epistasis was assessed using both stratified analysis and the linkage disequilibrium-based statistic. RESULTS: Mal S180L genotypes were similar in cases and controls (OR = 0.9, 95% CI 0.7 to 1.0, p = 0.2). Similarly, no difference for TLR4 G299D genotypes was seen (OR = 1.7, 95% CI 0.3 to 11.1, p = 0.5). No association with either rheumatoid factor or anti-cyclic citrullinated peptide status or with radiological damage was detected. Finally, no evidence of epistasis was detected between Mal S180L and TLR4 G299D and RA susceptibility. CONCLUSIONS: The Mal S180L and TLR4 G299D polymorphisms do not contribute to RA susceptibility or severity either individually or in combination.