Lifetime risk and genetic predisposition to post-traumatic OA of the knee in the UK Biobank.

OBJECTIVE: Acute knee injury is associated with post-traumatic OA (PTOA). Very little is known about the genome-wide associations of PTOA when compared with idiopathic OA (iOA). Our objective was to describe the development of knee OA after a knee injury and its genetic associations in UK Biobank (UKB). DESIGN: Clinically significant structural knee injuries in those ≤50 years were identified from electronic health records and self-reported data in 502,409 UKB participants. Time-to-first knee osteoarthritis (OA) code was compared in injured cases and age-/sex-matched non-injured controls using Cox Proportional Hazards models. A time-to-OA genome-wide association study (GWAS) sought evidence for PTOA risk variants 6 months to 20 years following injury. Evidence for associations of two iOA polygenic risk scores (PRS) was sought. RESULTS: Of 4233 knee injury cases, 1896 (44.8%) were female (mean age at injury 34.1 years [SD 10.4]). Over a median of 30.2 (IQR 19.5-45.4) years, 1096 (25.9%) of injured cases developed knee OA. The overall hazards ratio (HR) for knee OA after injury was 1.81 (1.70,1.93), P = 8.9 × 10-74. Female sex and increasing age at injury were associated with knee OA following injury (HR 1.15 [1.02,1.30];1.07 [1,07,1.07] respectively). OA risk was highest in the first 5 years after injury (HR 3.26 [2.67,3.98]), persisting for 40 years. In 3074 knee injury cases included in the time-to-OA GWAS, no variants reached genome-wide significance. iOA PRS was not associated with time-to-OA (HR 0.43 [0.02,8.41]). CONCLUSIONS: Increasing age at injury and female sex appear to be associated with future development of PTOA in UKB, the risk of which was greatest in the 5 years after injury. Further international efforts towards a better-powered meta-analysis will definitively elucidate genetic similarities and differences of PTOA and iOA.

Evidence for three genetic loci involved in both anorexia nervosa risk and variation of body mass index.

The maintenance of normal body weight is disrupted in patients with anorexia nervosa (AN) for prolonged periods of time. Prior to the onset of AN, premorbid body mass index (BMI) spans the entire range from underweight to obese. After recovery, patients have reduced rates of overweight and obesity. As such, loci involved in body weight regulation may also be relevant for AN and vice versa. Our primary analysis comprised a cross-trait analysis of the 1000 single-nucleotide polymorphisms (SNPs) with the lowest P-values in a genome-wide association meta-analysis (GWAMA) of AN (GCAN) for evidence of association in the largest published GWAMA for BMI (GIANT). Subsequently we performed sex-stratified analyses for these 1000 SNPs. Functional ex vivo studies on four genes ensued. Lastly, a look-up of GWAMA-derived BMI-related loci was performed in the AN GWAMA. We detected significant associations (P-values <5 × 10-5, Bonferroni-corrected P<0.05) for nine SNP alleles at three independent loci. Interestingly, all AN susceptibility alleles were consistently associated with increased BMI. None of the genes (chr. 10: CTBP2, chr. 19: CCNE1, chr. 2: CARF and NBEAL1; the latter is a region with high linkage disequilibrium) nearest to these SNPs has previously been associated with AN or obesity. Sex-stratified analyses revealed that the strongest BMI signal originated predominantly from females (chr. 10 rs1561589; Poverall: 2.47 × 10-06/Pfemales: 3.45 × 10-07/Pmales: 0.043). Functional ex vivo studies in mice revealed reduced hypothalamic expression of Ctbp2 and Nbeal1 after fasting. Hypothalamic expression of Ctbp2 was increased in diet-induced obese (DIO) mice as compared with age-matched lean controls. We observed no evidence for associations for the look-up of BMI-related loci in the AN GWAMA. A cross-trait analysis of AN and BMI loci revealed variants at three chromosomal loci with potential joint impact. The chromosome 10 locus is particularly promising given that the association with obesity was primarily driven by females. In addition, the detected altered hypothalamic expression patterns of Ctbp2 and Nbeal1 as a result of fasting and DIO implicate these genes in weight regulation.

Examining the overlap between genome-wide rare variant association signals and linkage peaks in rheumatoid arthritis.

OBJECTIVE: With the exception of the major histocompatibility complex (MHC) and STAT4, no other rheumatoid arthritis (RA) linkage peak has been successfully fine-mapped to date. This apparent failure to identify association under peaks of linkage could be ascribed to the examination of common variation, when linkage is likely to be driven by rare variants. The purpose of this study was to investigate the overlap between genome-wide rare variant RA association signals observed in the Wellcome Trust Case Control Consortium (WTCCC) study and 11 replicating RA linkage peaks, defined as regions with evidence for linkage in >1 study. METHODS: The WTCCC data set contained 40,482 variants with minor allele frequency of ≤0.05 in 1,860 RA patients and 2,938 controls. Genotypes of all rare variants within a given gene region were collapsed into a single locus and a global P value was calculated per gene. RESULTS: The distribution of rare variant signals (association P≤10(-5)) was found to differ significantly between regions with and without linkage evidence (P=2×10(-17) by Fisher's exact test). No significant difference was observed after data from the MHC region were removed or when the effect of the HLA-DRB1 locus was accounted for. CONCLUSION: The results suggest that rare variant association signals are significantly overrepresented under linkage peaks in RA, but the effect is driven by the MHC. This is the first study to examine the overlap between linkage peaks and rare variant association signals genome-wide in a complex disease.

An evaluation of power to detect low-frequency variant associations using allele-matching tests that account for uncertainty.

There is growing interest in the role of rare variants in multifactorial disease etiology, and increasing evidence that rare variants are associated with complex traits. Single SNP tests are underpowered in rare variant association analyses, so locus-based tests must be used. Quality scores at both the SNP and genotype level are available for sequencing data and they are rarely accounted for. A locus-based method that has high power in the presence of rare variants is extended to incorporate such quality scores as weights, and its power is compared with the original method via a simulation study. Preliminary results suggest that taking uncertainty into account does not improve the power.

Lack of significant effects of the type 2 diabetes susceptibility loci JAZF1, CDC123/CAMK1D, NOTCH2, ADAMTS9, THADA, and TSPAN8/LGR5 on diabetes and quantitative metabolic traits.

Recently, several novel loci reaching genome-wide significance levels for type 2 diabetes (T2D) were identified through a meta-analysis of three genome-wide scans and large-scale follow-up. The aim of our study was to investigate the association of these loci with T2D and related subphenotypes in two cohorts from Germany. We performed an association study of 9 SNPs in or around JAZF1, CDC123/ CAMK1D, NOTCH2, BCL11A, ADAMTS9, VEGFA, DCD, THADA, and TSPAN8/ LGR5 with T2D and related quantitative traits (fasting insulin and glucose, indices derived from OGTT) in the isolated population of Sorbs (205 cases and 695 controls) and in a mixed German population (Leipzig) (938 subjects with and 918 without T2D). None of the variants was associated with T2D, but the meta-analysis of both cohorts revealed a modest trend of association of rs7578597 in THADA with T2D (p=0.055). Furthermore, Sorbian subjects homozygous for the rs7578597 T-allele had lower mean 30-minute plasma insulin when compared with carriers of the C-allele (p<0.05). The T-allele was also nominally associated with higher fasting plasma glucose in the Leipzig cohort (p<0.05). Although several other SNPs showed some evidence for association with T2D-related traits the effects were not replicated within our study. Associations of the T2D-risk alleles with T2D or related subphenotypes were overall very weak in the approximately 2 700 subjects studied. This is compatible with the modest effect size of these "second sweep" variants, which will require large-scale association studies on quantitative traits to clarify their role in the pathophysiology of T2D.

Circulating beta-carotene levels and type 2 diabetes-cause or effect?

AIMS/HYPOTHESIS: Circulating beta-carotene levels are inversely associated with risk of type 2 diabetes, but the causal direction of this association is not certain. In this study we used a Mendelian randomisation approach to provide evidence for or against the causal role of the antioxidant vitamin beta-carotene in type 2 diabetes. METHODS: We used a common polymorphism (rs6564851) near the BCMO1 gene, which is strongly associated with circulating beta-carotene levels (p = 2 x 10(-24)), with each G allele associated with a 0.27 standard deviation increase in levels. We used data from the InCHIANTI and Uppsala Longitudinal Study of Adult Men (ULSAM) studies to estimate the association between beta-carotene levels and type 2 diabetes. We next used a triangulation approach to estimate the expected effect of rs6564851 on type 2 diabetes risk and compared this with the observed effect using data from 4549 type 2 diabetes patients and 5579 controls from the Diabetes Genetics Replication And Meta-analysis (DIAGRAM) Consortium. RESULTS: A 0.27 standard deviation increase in beta-carotene levels was associated with an OR of 0.90 (95% CI 0.86-0.95) for type 2 diabetes in the InCHIANTI study. This association was similar to that of the ULSAM study (OR 0.90 [0.84-0.97]). In contrast, there was no association between rs6564851 and type 2 diabetes (OR 0.98 [0.93-1.04], p = 0.58); this effect size was also smaller than that expected, given the known associations between rs6564851 and beta-carotene levels, and the associations between beta-carotene levels and type 2 diabetes. CONCLUSIONS/INTERPRETATION: Our findings in this Mendelian randomisation study are in keeping with randomised controlled trials suggesting that beta-carotene is not causally protective against type 2 diabetes.

Is the thrifty genotype hypothesis supported by evidence based on confirmed type 2 diabetes- and obesity-susceptibility variants?

AIMS/HYPOTHESIS: According to the thrifty genotype hypothesis, the high prevalence of type 2 diabetes and obesity is a consequence of genetic variants that have undergone positive selection during historical periods of erratic food supply. The recent expansion in the number of validated type 2 diabetes- and obesity-susceptibility loci, coupled with access to empirical data, enables us to look for evidence in support (or otherwise) of the thrifty genotype hypothesis using proven loci. METHODS: We employed a range of tests to obtain complementary views of the evidence for selection: we determined whether the risk allele at associated 'index' single-nucleotide polymorphisms is derived or ancestral, calculated the integrated haplotype score (iHS) and assessed the population differentiation statistic fixation index (F (ST)) for 17 type 2 diabetes and 13 obesity loci. RESULTS: We found no evidence for significant differences for the derived/ancestral allele test. None of the studied loci showed strong evidence for selection based on the iHS score. We find a high F (ST) for rs7901695 at TCF7L2, the largest type 2 diabetes effect size found to date. CONCLUSIONS/INTERPRETATION: Our results provide some evidence for selection at specific loci, but there are no consistent patterns of selection that provide conclusive confirmation of the thrifty genotype hypothesis. Discovery of more signals and more causal variants for type 2 diabetes and obesity is likely to allow more detailed examination of these issues.

Gene variants influencing measures of inflammation or predisposing to autoimmune and inflammatory diseases are not associated with the risk of type 2 diabetes.

AIMS/HYPOTHESIS: There are strong associations between measures of inflammation and type 2 diabetes, but the causal directions of these associations are not known. We tested the hypothesis that common gene variants known to alter circulating levels of inflammatory proteins, or known to alter autoimmune-related disease risk, influence type 2 diabetes risk. METHODS: We selected 46 variants: (1) eight variants known to alter circulating levels of inflammatory proteins, including those in the IL18, IL1RN, IL6R, MIF, PAI1 (also known as SERPINE1) and CRP genes; and (2) 38 variants known to predispose to autoimmune diseases, including type 1 diabetes. We tested the associations of these variants with type 2 diabetes using a meta-analysis of 4,107 cases and 5,187 controls from the Wellcome Trust Case Control Consortium, the Diabetes Genetics Initiative, and the Finland-United States Investigation of NIDDM studies. We followed up associated variants (p < 0.01) in a further set of 3,125 cases and 3,596 controls from the UK. RESULTS: We found no evidence that inflammatory or autoimmune disease variants are associated with type 2 diabetes (at p

Disparate genetic influences on polycystic ovary syndrome (PCOS) and type 2 diabetes revealed by a lack of association between common variants within the TCF7L2 gene and PCOS.

AIMS/HYPOTHESIS: Common variants of the gene encoding transcription factor 7-like 2 (TCF7L2) have a powerful effect on individual risk of type 2 diabetes (per allele odds ratio approximately 1.35). Polycystic ovary syndrome (PCOS) and type 2 diabetes are familial conditions sharing common features. Based on this, the aim of the present study was to establish whether variation in TCF7L2 also influences the development of PCOS. METHODS: We conducted a genetic association study of variants of TCF7L2 (rs7903146 and rs12255372) using both case-control and quantitative trait approaches. Case-control analyses were conducted in (1) 369 PCOS cases and 2574 controls of UK British/Irish origin, and (2) 540 women with PCOS symptoms and 1083 controls from the Northern Finland Birth Cohort of 1966. Quantitative trait analyses (androgen levels) were also performed (1249 individuals). RESULTS: There was no association between rs7903146 and PCOS in the UK case-control study (Cochran-Armitage test, p = 0.51); nor with symptomatic status in the Finnish cohort (p = 0.36). In addition, there were no relationships between the TCF7L2 single nucleotide polymorphism rs7903146 and androgen levels (UK cases, p = 0.99; Finnish controls, p = 0.57; Finnish symptomatic cases, p = 0.80). Results at rs12255372 were similar, reflecting strong linkage disequilibrium with rs7903146. CONCLUSIONS/INTERPRETATION: Our study was powered to detect an effect on PCOS susceptibility similar to that previously reported for these variants on type 2 diabetes. Failure to detect any evident association with PCOS provides the strongest evidence yet that the genetic architecture of these related conditions is qualitatively distinct.

An association analysis of the HLA gene region in latent autoimmune diabetes in adults.

AIMS/HYPOTHESIS: Pathophysiological similarities between latent autoimmune diabetes in adults (LADA) and type 1 diabetes indicate an overlap in genetic susceptibility. HLA-DRB1 and HLA-DQB1 are major susceptibility genes for type 1 diabetes but studies of these genes in LADA have been limited. Our aim was to define patterns of HLA-encoded susceptibility/protection in a large, well characterised LADA cohort, and to establish association with disease and age at diagnosis. MATERIALS AND METHODS: Patients with LADA (n = 387, including 211 patients from the UK Prospective Diabetes Study) and non-diabetic control subjects (n = 327) were of British/Irish European origin. The HLA-DRB1 and -DQB1 genes were genotyped by sequence-specific PCR. RESULTS: As in type 1 diabetes mellitus, DRB1 0301_DQB1 0201 (odds ratio [OR] = 3.08, 95% CI 2.32-4.12, p = 1.2 x 10(-16)) and DRB1 0401_DQB1 0302 (OR = 2.57, 95% CI 1.80-3.73, p = 4.5 x 10(-8)) were the main susceptibility haplotypes in LADA, and DRB1 1501_DQB1 0602 was protective (OR = 0.21, 95% CI 0.13-0.34, p = 4.2 x 10(-13)). Differential susceptibility was conferred by DR4 subtypes: DRB1 0401 was predisposing (OR = 1.79, 95% CI 1.35-2.38, p = 2.7 x 10(-5)) whereas DRB1 0403 was protective (OR = 0.37, 95% CI 0.13-0.97, p = 0.033). The highest-risk genotypes were DRB1 0301/DRB1 0401 and DQB1 0201/DQB1 0302 (OR = 5.14, 95% CI 2.68-10.69, p = 1.3 x 10(-8); and OR = 6.88, 95% CI 3.54-14.68, p = 1.2 x 10(-11), respectively). These genotypes and those containing DRB1 0401 and DQB1 0302 associated with a younger age at diagnosis in LADA, whereas genotypes containing DRB1 1501 and DQB1 0602 associated with an older age at diagnosis. CONCLUSIONS/INTERPRETATION: Patterns of susceptibility at the HLA-DRB1 and HLA-DQB1 loci in LADA are similar to those reported for type 1 diabetes, supporting the hypothesis that autoimmune diabetes occurring in adults is an age-related extension of the pathophysiological process presenting as childhood-onset type 1 diabetes.

Paraoxonase 2 (PON2) polymorphisms and development of renal dysfunction in type 2 diabetes: UKPDS 76.

AIMS/HYPOTHESIS: Identification of variants predicting development of renal dysfunction would offer substantial clinical benefits. There is evidence that coding non-synonymous variants in the gene encoding paraoxonase 2 (PON2) are associated with nephropathy in both type 1 and type 2 diabetes. METHODS: We examined the relationship between variation at the C311S and A148G polymorphisms (together with PON2 intronic variant rs12704795) and indices of renal dysfunction (progression to micro- and macroalbuminuria, plasma creatinine increases) in 3,374 newly diagnosed type 2 diabetic subjects from the UK Prospective Diabetes Study followed prospectively (median 14.0 years), using proportional hazards models, adjusted for sex, ethnicity and other known or putative risk factors. RESULTS: rs12704795 genotypes were associated with differing rates of development of microalbuminuria (relative risk [RR] for CC vs AA homozygotes 0.68 [95% CI 0.54-0.87], p=0.002) but not other measures of worsening renal function. Heterozygotes for C311S were more likely to develop microalbuminuria (RR=1.31 [95% CI 1.11-1.54], p=0.001) but less likely to double creatinine levels during follow-up (RR=0.49 [95% CI 0.27-0.89], p=0.02). There was no corroboration of this latter association for related outcomes and no prior evidence supports heterosis effects at this locus. CONCLUSIONS/INTERPRETATION: We conclude that the PON2 variants typed in this study have, at best, a small effect on the risk of renal dysfunction in type 2 diabetes.

Association of HLA-DRB1*13 with susceptibility to uveitis in juvenile idiopathic arthritis in two independent data sets.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is the commonest rheumatic disease of childhood. Uveitis is the commonest eye complication of JIA, potentially leading to eye surgery and/or visual loss. JIA is a complex genetic trait with well-established HLA-DRB1 associations. The aim of this study was to investigate the involvement of HLA-DRB1 in JIA-associated uveitis. METHODS: A set of 130 UK Caucasian simplex families consisting of healthy parent(s) and a child affected with juvenile oligoarticular idiopathic arthritis (of which 31 had developed uveitis) had previously been screened for multiple markers in the major histocompatibility complex region. Associations with uveitis were investigated through haplotype pattern mining (HPM) and the extended transmission disequilibrium test (ETDT). A further set of 228 UK Caucasian patients with long-standing JIA were fully genotyped for HLA-DRB1 using PCR with sequence-specific primers. Associations of HLA-DRB1 alleles in patients with uveitis (n = 50) were examined individually using the chi 2 test. RESULTS: In the first cohort, HPM identified significant associations of HLA-DRB1*13 with uveitis in juvenile oligoarthritis (P = 0.002). The ETDT confirmed overtransmission of this allele in the families (empirical global P = 0.018). In the second cohort, the significant association of uveitis with HLA-DRB1*13 was replicated (P = 0.0002, odds ratio 3.4, 95% confidence interval 1.7-6.5). CONCLUSIONS: This study has established the HLA-DRB1*13 association with uveitis in JIA. Further work is necessary in order to explore the prognostic potential of this marker.

Large-scale studies of the association between variation at the TNF/LTA locus and susceptibility to type 2 diabetes.

AIMS/HYPOTHESIS: The proinflammatory cytokine TNF-alpha has been implicated in the pathogenesis of insulin resistance and type 2 diabetes, and variation in the gene encoding TNF-alpha (TNF) has shown inconsistent associations with susceptibility to both conditions. Additionally, the coding non-synonymous variant T60N in the neighbouring LTA gene has been reported to be associated with type 2 diabetes. The present study aimed to obtain a robust assessment of the role of variation in the tightly linked TNF/LTA region in diabetes susceptibility by genotyping TNF and LTA variants in large case-control resources. MATERIALS AND METHODS: The G-308A and G-238A TNF promoter variants and the LTA T60N polymorphism were genotyped in two UK case samples that were ascertained for positive family history and/or early onset of type 2 diabetes (combined n=858) and in 1,257 ethnically matched controls. RESULTS: There were no significant associations between the T60N, G-308A or G-238A genotype and type 2 diabetes in the combined analysis (exact Cochran-Mantel-Haenszel statistic for ordered genotypes for T60N, p=0.69; for G-308A, p=0.51; for G-238A, p=0.16). CONCLUSIONS/INTERPRETATION: The present study, one of the largest association analyses yet reported at this locus, provides no evidence that the specific TNF or LTA variants examined influence susceptibility to type 2 diabetes. More comprehensive studies of the TNF/LTA locus in substantially larger sample sets are required to establish whether genome sequence variation at this locus truly influences susceptibility to type 2 diabetes.

The type 1 diabetes susceptibility gene SUMO4 at IDDM5 is not associated with susceptibility to rheumatoid arthritis or juvenile idiopathic arthritis.

OBJECTIVES: Linkage and association of rheumatoid arthritis (RA) and rheumatoid factor (RF)-negative juvenile idiopathic arthritis (JIA) has previously been demonstrated to the type 1 diabetes (T1D) locus, IDDM5, on chromosome 6q25. An association of a methionine-to-valine polymorphism (rs237025, 163A --> G, M55V) in the SUMO4 gene within IDDM5 has recently been described in T1D. The objective of this study was to test the hypothesis that SUMO4 is a general autoimmune susceptibility gene by investigating whether the SUMO4 polymorphism is associated with RA and/or JIA. METHODS: The SUMO4 SNP was genotyped in 875 RA patients, 668 JIA patients and 484 healthy controls using a TaqMan allelic discrimination assay. Allele and genotype frequencies were compared between cases and controls using the chi2 test. Analyses were also carried out with RA patients stratified by gender, age at onset, RF status, the presence of erosive disease and shared epitope status, while JIA patients were stratified by their International League of Associations for Rheumatology (ILAR) subgroup. RESULTS: No deviation from Hardy-Weinberg equilibrium was detected in either set of cases or controls. No association was observed between rs237025 and RA (chi2 = 0.17, P = 0.93), or with any RA subset. Similarly, there was no association between this SNP and JIA (chi2 = 0.21, P = 0.90), or with any ILAR subgroup. CONCLUSIONS: The M55V substitution in the SUMO4 gene is not associated with susceptibility to RA or JIA in the UK population studied. However, other candidate genes mapping within IDDM5 remain to be investigated.

Examining the relationships between the Pro12Ala variant in PPARG and Type 2 diabetes-related traits in UK samples.

AIMS: The Pro12Ala polymorphism in the PPARG gene alters amino acid sequence and has shown consistent association with susceptibility to Type 2 diabetes in several populations. The present study makes use of large, well-characterized case-control resources to enhance understanding of this susceptibility effect by examining related traits, such as body mass index (BMI), waist-hip ratio and age at diagnosis. METHODS: The Pro12Ala variant was genotyped in two UK case samples, ascertained for positive family history and/or early onset of Type 2 diabetes (combined n=971); and in 1257 ethnically matched control subjects. RESULTS: There were significant associations of the Pro12Ala single nucleotide polymorphism (SNP) genotypes with diabetes in both case-control comparisons (P=0.025 and P=0.039). Comparing individuals homozygous for the Pro allele, with those carrying an Ala allele, the combined odds ratio for diabetes was 1.40 (95% CIs, 1.12-1.76, P=0.0031). There was no association between the variant and either waist-hip ratio or age at diagnosis. Proline homozygosity was associated with increased BMI in one patient group (P=0.013) and decreased BMI in the other (P=0.038). CONCLUSIONS: This study confirms that variation within PPARG influences susceptibility to Type 2 diabetes in UK samples. However, the relationship between PPARG variation and BMI is more complex, and studies in much larger sample sets will be required to more precisely characterize the effect of this variant on adiposity.

Toll-like receptor 4 gene polymorphisms and susceptibility to juvenile idiopathic arthritis.

OBJECTIVES: To determine if polymorphisms within the Toll-like receptor 4 (TLR4) gene are associated and linked with juvenile idiopathic arthritis (JIA). To investigate any possible gene-gene (epistatic) interaction between TLR4 and macrophage migration inhibitory factor (MIF) gene polymorphisms. METHODS: 313 simplex families (each containing one affected JIA proband) were genotyped. Two known functionally important single nucleotide polymorphisms (SNPs) within the TLR4 gene (Asp299Gly and Thr399Ile) were typed by SNaPshot ddNTP primer extension and capillary electrophoresis. Single point and multipoint transmission disequilibrium tests (TDT) were carried out through the extended TDT and TDT phase packages for the two TLR4 SNPs. Epistatic interaction between TLR4 haplotypes and the previously JIA associated MIF CATT(7)-MIF-173*C promoter haplotype was investigated by chi(2) test and unconditional logistic regression in Stata version 7. RESULTS: No distortion from random inheritance was observed by single point analysis for TLR4 Asp299Gly (p = 0.89) or TLR4 Thr399Ile (p = 0.40). Similarly, no distortion in transmission was seen when the TLR4 haplotypes were studied (p = 0.54). Additionally, no evidence for gene-gene interaction between TLR4 polymorphisms and the previously associated MIF gene polymorphisms was found (p = 0.40). CONCLUSIONS: No linkage or association was seen for Asp299Gly or Thr399Ile SNPs of TLR4 with JIA susceptibility. No evidence of an epistatic interaction between these TLR4 polymorphisms and MIF polymorphisms was found.