Genetically determined high activities of the TNF-alpha, IL23/IL17, and NFkB pathways were associated with increased risk of ankylosing spondylitis

Background Ankylosing spondylitis (AS) results from the combined effects of susceptibility genes and environmental factors. Polymorphisms in genes regulating inflammation may explain part of the heritability of AS. Methods Using a candidate gene approach in this case-control study, 51 mainly functional single nucleotide polymorphisms (SNPs) in genes regulating inflammation were assessed in 709 patients with AS and 795 controls. Data on the patients with AS were obtained from the DANBIO registry where patients from all of Denmark are monitored in routine care during treatment with conventional and biologic disease modifying anti-rheumatic drugs (bDMARDs). The results were analyzed using logistic regression (adjusted for age and sex). Results Nine polymorphisms were associated with risk of AS (p < 0.05). The polymorphisms were in genes regulating a: the TNF-α pathway (TNF -308 G > A (rs1800629), and − 238 G > A (rs361525); TNFRSF1A -609 G > T (rs4149570), and PTPN22 1858 G > A (rs2476601)), b: the IL23/IL17 pathway (IL23R G > A (rs11209026), and IL18–137 G > C (rs187238)), or c: the NFkB pathway (TLR1 743 T > C (rs4833095), TLR4 T > C (rs1554973), and LY96–1625 C > G (rs11465996)). After Bonferroni correction the homozygous variant genotype of TLR1 743 T > C (rs4833095) (odds ratios (OR): 2.59, 95% confidence interval (CI): 1.48–4.51, p = 0.04), and TNFRSF1A -609 G > T (rs4149570) (OR: 1.79, 95% CI: 1.31–2.41, p = 0.01) were associated with increased risk of AS and the combined homozygous and heterozygous variant genotypes of TNF -308 G > A (rs1800629) (OR: 0.56, 95% CI: 0.44–0.72, p = 0.0002) were associated with reduced risk of AS. Conclusion We replicated associations between AS and the polymorphisms in TNF (rs1800629), TNFRSF1A (rs4149570), and IL23R (rs11209026). Furthermore, we identified novel risk loci in TNF (rs361525), IL18 (rs187238), TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996) that need validation in independent cohorts. The results suggest that genetically determined high activity of the TNF-α, IL23/IL17, and NFkB pathways increase risk of AS.


Background
Ankylosing spondylitis (AS) is a type of spondyloarthritis in which hallmark clinical features are inflammation at entheses and subchondral bone of the pelvic and spinal joints with subsequent abnormal new bone formation at these sites. Ultimately, this leads to ossification of entheses and joints resulting in loss of joint mobility. The incidence varies between 0.1 and 1.8% with the highest incidence in Scandinavia. Onset is typically in young adults with a male predominance. Medications used include non-steroid anti-inflammatory drugs (NSAIDs), and biological diseasemodifying anti-rheumatic drugs (bDMARDs), i.e. tumor necrosis factor-α inhibitors (anti-TNF) and more recently also an interleukin(IL)-17A inhibitor (secukinumab) [1].
The cause of AS is unknown but is believed to involve a combination of genetic and environmental factors [2]. The heritability is polygenic and estimated to exceed 90%, with the HLA-B27 allele as the major contributor accounting for approximately 25% of the heritability of AS [2]. The IL-17/ IL-23 pathway and the TNF-α pathway are central in the pathogenesis of AS and alterations in these pathways have been shown in mouse models to affect development and severity of enthesitis [3,4].
TNF-α can be activated by Pathogen-Associated Molecular Patterns (PAMPs) such as bacterial or viral DNA, flagellin, or lipopolysaccharide (LPS), through the NFkB pathway. PAMPs can be recognized by Toll-like receptors (TLRs) thereby initiating a kinase cascade which phosphorylates and degrades the NFkB inhibitor IkBα [5]. This releases NFkB which is transported from the cytosol to the nucleus where it initiates expression of pro-and anti-inflammatory cytokines including TNF-α and IL-17 (http://www.bu.edu/nf-kb/gene-resources/targetgenes/). The TNF-α and NFkB pathway are intertwined and TNF-α can feedback stimulate NFkB by binding to TNF receptors (TNFR1 or TNFR2), resulting in a kinase cascade similar to, but distinct from, the pathway induced by TLRs [5].
The IL23/IL17 pathway can also stimulate TNF-α activity. The pro-inflammatory cytokine IL-17 enhances the production of other pro-inflammatory cytokines including TNF-α, and the secretion IL-17 itself can be enhanced by IL-23 [6].
The aim of this study was to assess whether functional single nucleotide polymorphisms.
(SNPs) in genes involved in the TNF-α, IL23/IL17, NFkB, and other pro-and anti-inflammatory pathways were associated with risk of AS.

Patients and samples
The DANBIO registry includes prospectively collected clinical data on patients with inflammatory joint diseases including smoking status, disease characteristics e.g. HLA-B27 status, disease activity, treatment, and treatment outcomes. Patients from all of Denmark are monitored in routine care during treatment with conventional and biologic disease modifying anti-rheumatic drugs (bDMARDs) [9].
Screening for tuberculosis before initiation of treatment with biological drugs is routinely performed in Denmark. Left over blood clots (after whole blood analysis for Mycobacterium tuberculosis) were collected from all patients screened for tuberculosis at Statens Serum Institut (Copenhagen, Denmark) from 01. 09 By linking the unique personal identification number of Danish citizens (CPR-number) from each blood sample with the clinical data from DANBIO, 709 patients with AS (ICD-10: M45.9) were identified. The control group consisted of 795 healthy blood donors recruited from Viborg, Denmark.

Power calculation
The Genetic Power Calculator was utilized for power analysis of discrete traits (http://zzz.bwh.harvard.edu/ gpc/cc2.html). The lowest minor allele frequency (MAF) of the studied SNPs was 0.10. The 'high-risk allele frequency' was set to 0.10, the 'prevalence' was set to 0.0018 [18], D-prime was set to 1, type I error rate was set to 0.05 and number of cases and control:case ratio was 795:709. This cohort study had more than 80% chance of detecting a dominant effect with an odds ratio (OR) of 1.4 for AS.

Statistical analysis
Logistic regression was used to compare genotype distributions among patients with AS versus healthy controls. Crude odds ratio, odds ratio adjusted for age and sex, and odds ratio adjusted for age, sex, and smoking status were assessed (Table 1). A chi-square test was
SNPs associated with AS and the biological effect of the SNPs are summarized in Table 2.
The TLR4 haplotype combination 33 (rs12377632TT, rs1554973CC and rs5030728GG) was associated with reduced risk of AS (OR: 0.54, 95% CI: 0.32-0.92, p = 0.03) compared to the haplotype combination 11. In TNF all haplotype combinations were associated with reduced risk of AS compared to the haplotype combination 11 (rs361525GG and rs1800629GG).
No associations were found for haplotype combinations of TLR2 or IL1B.  The variant allele of TNF -238A rs361525A G > A has been shown to reduce expression of TNF-α [49] c The variant allele of TNF -308A rs1800629 G > A has been shown to reduce mRNA level [48]
Most of the SNPs assessed in our study have known biological effects thus allowing a biological interpretation of the observed associations based on increased or reduced gene activity as summarized in Table 2 [35][36][37][38][39][40][41][42][43][44][45][46][47]. The associations observed for the TNF (rs1800629 and rs361525) polymorphisms suggest that reduced TNF-α mRNA level and expression of TNF-α was associated with reduced risk of AS [48,49]. This is supported by our haplotype analysis which also suggests that the variant alleles of TNF rs1800629 and rs361525 were associated with reduced risk of AS. Likewise, the associations observed for the TNFRSF1A (rs4149570) polymorphism indicates that increased expression of the TNF-α receptor 1 was associated with increased risk of AS [50]. Furthermore, the associations observed for the PTPN22 (rs2476601) polymorphism suggests that reduced TNF-α serum level was associated with reduced risk of AS [51]. Taken together, this suggests that genetically determined high activity of the TNF-α pathway was associated with increased risk of AS.
IL-17 is known to induce the production of many cytokines including TNF-α [6]. IL-18 is a pro-inflammatory cytokine known to enhance the production of IL-17, TNF-α, and IL-1β [8]. In this study, the association observed for the IL23R (rs11209026) polymorphism suggests that reduced IL-17 serum level, and thus reduced TNF-α activity, was associated with reduced risk of AS [52]. Furthermore, the associations observed for the IL18 (rs187238) polymorphism indicates that reduced IL-18 expression, and thus reduced IL-17 and TNF-α activity, was associated with reduced risk of AS [53,54]. The associations found in the IL23R (rs11209026) and the IL18 (rs187238) polymorphisms thus suggest that a genetically determined high activity of the IL23/IL17 pathway was associated with increased risk of AS. The two SNPs furthermore support that genetically determined high activity of the TNF-α pathway was associated with increased risk of AS. The observed associations between the polymorphisms in IL23R and IL18 and risk of AS are in line with previous studies pointing out the IL23/IL17 pathway as central to the pathophysiology of AS [3,4,55].
This study also suggests that the NFkB pathway may be involved in the etiology of AS. The associations observed for the TLR1 (rs4833095) polymorphism suggests that increased TLR1 level was associated with increased risk of AS [56]. High level of TLR1 may lead to increased NFkB activation and thus increased TNF-α and IL-17 activity, which is in line with the other results. However, in contrast to the other results, the associations observed for the LY96 (rs11465996) polymorphism suggests that increased MD-2 (LY96) and TNF-α level was associated with a reduced risk of AS [57]. Finally, the TLR4 (rs1554973) polymorphism was associated with reduced risk of AS which was supported by the haplotype results ( Table 4). The biological effect of the TLR4 (rs1554973) polymorphism is unknown, however, the result supports the notion that the NFkB pathway may be involved in the etiology of AS.
There are aspects of this study which should be interpreted with care. Conflicting results have been reported for the TNF (rs1800629) polymorphism [48,49,65]. Furthermore, the TNF polymorphisms, as well as the HLA-B27 locus, are located on chromosome 6, and there is a risk that even a minor linkage disequilibrium could have confounded our results [2]. TLR1 (rs4833095), TLR2 (rs4696480), TLR4 (rs1554973), TLR9 (rs352139), and TGFB1 (rs1800469) were not in Hardy-Weinberg equilibrium among the healthy controls. Due to the number of polymorphisms analyzed this is probably a type II error. The polymorphisms do not deviate from Hardy-Weinberg equilibrium when corrected for multiple testing. We cannot exclude that some of our positive findings may be due to chance due to the obtained p-values and the number of statistical tests performed. When the results were corrected for multiple testing only the variant allele of TLR1 (rs4833095) and TNFRSF1A (rs4149570) were associated with increased risk of AS and the variant allele of TNF (rs1800629) was associated with reduced risk of AS.
A major strength of this study was that the cohort was rather large including 709 patients with AS and 795 healthy controls and the associations that we report were biologically plausible. Also, the validity of the diagnosis is expected to be high, since the patients were identified via a clinical database that the rheumatologist use for prospective monitoring of patients as part of routine care [66].

Conclusions
In conclusion, we replicated associations between AS and the polymorphism TNF (rs1800629), TNFRSF1A (rs4149570), and IL23R (rs11209026). Furthermore, we identified novel risk loci in TNF (rs361525), IL18 (rs18 7238), TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996) that need validation in independent cohorts. The results suggest that genetically determined high activity of the TNF-α, IL23/IL17, and NFkB pathways increase the risk of AS. Co-author Niels H.H. Heegaard, Professor, MD, DMSc, DNatSc, died unexpectedly on September 26, 2017, at age 57. As director of the Department of Autoimmunology and Biomarkers, Statens Serum Institut, Copenhagen, Dr. Heegaard advanced research in autoimmunology and neurodegenerative disease. He had an extensive international research network and published more than 200 papers in scientific journals, focusing on biomarkers such as autoantibodies, microRNA, and microparticle proteins. He was a patient and unpretentious collaborator who always sought to highlight the work of other collaborators and co-workers. Dr. Heegaard was characterized by humor, kindness, and optimism. He is survived by his wife and 2 children.

Availability of data and materials
The datasets used during the current study are available from the corresponding author on reasonable request.
Authors' contributions JS, SB, UV, PSA, SBS, HL, NHH and VA designed the research study and PSA, ABB, MRA, IB, RBD, HJH, BG and MLH collected the materials. JS and SB analysed the data and wrote the first draft. UV, PSA, SBS, ABB, MRA, IB, RBD, HJH, BG, MLH, HL and VA critically revised the manuscript. All authors agreed to be accountable for all aspects of the work and approved the final version of the manuscript.

Ethics approval and consent to participate
The study was conducted in accordance with the Declaration of Helsinki and was approved by the Regional Ethics Committees of Central (M20100153) and Southern (S-20120113) Denmark and the Danish Data Protection Agency of Central (RM: J. 2010-41-4719) and Southern (RSD: 2008-58-035) Denmark. For blood samples collected after routine TB screening, the Ethics Committees gave exemption from informed consent requirements because samples were taken as part of routine care and data were not identifiable. Written informed consent was obtained from patients donating blood samples at Frederiksberg Hospital as this involved collecting additional samples from patients.

Consent for publication Not applicable.
Competing interests VA receives compensation as a consultant and for being member of an advisory board for MSD and Janssen. BG has recived research funding from AbbVie, Biogen, Pfizer. The other authors declare no conflicts of interest.

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