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. 2018 Sep 12;19:165. doi: 10.1186/s12881-018-0680-z

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

Jacob Sode 1,2,3,4, Steffen Bank 5,6,✉, Ulla Vogel 7, Paal Skytt Andersen 8,9, Signe Bek Sørensen 1,5,10, Anders Bo Bojesen 5, Malene Rohr Andersen 11, Ivan Brandslund 12, Ram Benny Dessau 13, Hans Jürgen Hoffmann 14,15, Bente Glintborg 16,17, Merete Lund Hetland 17,18, Henning Locht 3, Niels Henrik Heegaard 2,19, Vibeke Andersen 1,5,10,20
PMCID: PMC6136164  PMID: 30208882

Abstract

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.

Keywords: Ankylosing spondylitis, Single nucleotide polymorphism, SNP, Case-control study

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 disease-modifying 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/target-genes/). 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].

PAMPs can also be recognized by intracellular Nod-like receptors (NLRs). In turn, NLRs can activate pro-inflammatory cytokines including IL-18 [7]. IL-18 is invloved in the IL23/IL17 pathway and can enhance the production of IL-17 [8].

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.

Methods

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 01.09.2009 to 31.01.2013; the Department of Respiratory Diseases B and the Department of Clinical Microbiology, Aarhus University Hospital (Aarhus, Denmark) from 01.01.2011 to 31.01.2014; the Department of Clinical Biochemistry, Herlev and Gentofte Hospital (Hellerup, Denmark) from 01.03.2012 to 31.01.2014; the Department of Biochemistry, Hospital of Lillebaelt (Vejle, Denmark); and the Department of Biochemistry, Hospital of Slagelse (Slagelse, Denmark) from 01.01.2014 to 31.01.2014. Furthermore, from 01.01.2013 to 31.12.2013 blood samples were collected from all patients with AS treated with or without anti-TNF drugs at the Department of Rheumatology, Frederiksberg Hospital (Frederiksberg, Denmark).

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.

Genotyping

Fifty-one SNPs in genes involved in the TNF-α, IL23/IL17, NFκB, and other pro- and anti-inflammatory pathways were assessed. A list of all SNPs studied and genotype distribution is presented in Table 1 and SNPs associated with AS are summarized in Table 2.

Table 1.

Odds ratios (OR) and 95% confidence interval (95CI) for genotypes studied among healthy controls and patients with ankylosing spondylitis (AS)

Gene
rs-number		 Healthy controls AS Unadjusted Adjusted, age & sex Adjusted, age, sex & smoking
OR (95 CI) p OR (9 5CI) p OR (95 CI) p
TLR1
rs4833095
 TT 485 415
 TC 261 238 1.07 (0.86–1.33) 0.57 1.03 (0.82–1.29) 0.83 1.05 (0.78–1.42) 0.73
 CC 20 43 2.51 (1.45–4.34) 0.00095 2.59 (1.48–4.51) 0.00081 2.86 (1.44–5.68) 0.0026
 TC or CC 281 281 1.17 (0.95–1.44) 0.15 1.14 (0.91–1.41) 0.25 1.18 (0.89–1.58) 0.26
TLR2
rs3804099
 TT 241 197
 TC 393 354 1.10 (0.87–1.40) 0.42 1.07 (0.84–1.37) 0.58 1.02 (0.73–1.42) 0.90
 CC 144 142 1.21 (0.89–1.63) 0.22 1.24 (0.91–1.68) 0.17 1.30 (0.87–1.96) 0.20
 TC or CC 537 496 1.13 (0.90–1.41) 0.29 1.11 (0.89–1.40) 0.36 1.10 (0.80–1.50) 0.57
TLR2
rs11938228
 CC 327 314
 CA 368 313 0.89 (0.71–1.10) 0.27 0.86 (0.69–1.07) 0.17 0.80 (0.60–1.08) 0.15
 AA 76 69 0.95 (0.66–1.36) 0.76 0.92 (0.63–1.33) 0.66 1.03 (0.62–1.69) 0.92
 CA or AA 444 382 0.90 (0.73–1.10) 0.30 0.87 (0.70–1.07) 0.19 0.84 (0.63–1.11) 0.22
TLR2
rs4696480
 AA 199 179
 AT 417 348 0.93 (0.72–1.19) 0.55 0.89 (0.69–1.15) 0.38 0.84 (0.60–1.18) 0.31
 TT 155 169 1.21 (0.90–1.63) 0.20 1.16 (0.86–1.58) 0.33 1.18 (0.78–1.78) 0.44
 AT or TT 572 517 1.00 (0.79–1.27) 0.97 0.97 (0.76–1.23) 0.78 0.92 (0.67–1.27) 0.62
TLR4
rs5030728
 GG 359 322
 GA 323 298 1.03 (0.83–1.28) 0.80 1.01 (0.81–1.27) 0.91 0.93 (0.69–1.25) 0.62
 AA 78 70 1.00 (0.70–1.43) 1.00 0.98 (0.68–1.42) 0.93 0.87 (0.53–1.42) 0.57
 GA or AA 401 368 1.02 (0.83–1.26) 0.83 1.01 (0.82–1.25) 0.94 0.91 (0.69–1.21) 0.53
TLR4
rs1554973
 TT 440 395
 TC 272 261 1.07 (0.86–1.33) 0.55 1.06 (0.85–1.32) 0.62 0.98 (0.73–1.32) 0.90
 CC 62 33 0.59 (0.38–0.92) 0.02 0.55 (0.34–0.86) 0.01 0.68 (0.38–1.23) 0.20
 TC or CC 334 294 0.98 (0.80–1.21) 0.85 0.96 (0.78–1.19) 0.72 0.93 (0.70–1.24) 0.63
TLR4
rs12377632
 TT 306 271
 TC 358 319 1.01 (0.81–1.26) 0.96 1.05 (0.84–1.32) 0.66 1.07 (0.78–1.46) 0.67
 CC 102 96 1.06 (0.77–1.47) 0.71 1.11 (0.80–1.55) 0.52 1.41 (0.92–2.17) 0.12
 TC or CC 460 415 1.02 (0.83–1.26) 0.86 1.06 (0.86–1.32) 0.58 1.14 (0.85–1.53) 0.37
TLR5
rs5744168
 CC 672 605
 CT 94 89 1.05 (0.77–1.43) 0.75 1.05 (0.77–1.45) 0.74 0.89 (0.58–1.37) 0.60
 TT 5 2 0.44 (0.09–2.30) 0.33 0.45 (0.08–2.43) 0.35 0.04 (0.00–3.54) 0.16
 CT or TT 99 91 1.02 (0.75–1.39) 0.89 1.02 (0.75–1.40) 0.88 0.84 (0.55–1.29) 0.43
TLR5
rs5744174
 TT 215 216
 TC 399 337 0.84 (0.66–1.07) 0.15 0.85 (0.67–1.09) 0.20 0.82 (0.60–1.14) 0.24
 CC 144 138 0.95 (0.71–1.29) 0.76 1.02 (0.75–1.39) 0.91 0.87 (0.57–1.32) 0.51
 TC or CC 543 475 0.87 (0.69–1.09) 0.23 0.90 (0.71–1.13) 0.36 0.84 (0.62–1.14) 0.26
TLR9
rs187084
 TT 262 237
 TC 366 335 1.01 (0.80–1.27) 0.92 1.03 (0.82–1.31) 0.78 1.09 (0.79–1.50) 0.60
 CC 142 120 0.93 (0.69–1.26) 0.66 0.91 (0.67–1.24) 0.56 1.07 (0.71–1.61) 0.76
 TC or CC 508 455 0.99 (0.80–1.23) 0.93 1.00 (0.80–1.25) 0.98 1.08 (0.80–1.46) 0.60
TLR9
rs352139
 GG 255 211
 GA 347 324 1.13 (0.89–1.43) 0.32 1.08 (0.85–1.38) 0.52 1.01 (0.73–1.40) 0.93
 AA 167 139 1.01 (0.75–1.34) 0.97 0.96 (0.71–1.30) 0.79 0.80 (0.53–1.20) 0.27
 GA or AA 514 463 1.09 (0.87–1.36) 0.45 1.04 (0.83–1.31) 0.72 0.94 (0.69–1.27) 0.68
LY96
rs11465996
 CC 344 341
 CG 337 298 0.89 (0.72–1.11) 0.30 0.91 (0.73–1.14) 0.42 0.89 (0.66–1.20) 0.45
 GG 81 53 0.66 (0.45–0.96) 0.03 0.68 (0.46–1.00) 0.0498 0.65 (0.39–1.10) 0.11
 CG or GG 418 351 0.85 (0.69–1.04) 0.11 0.87 (0.70–1.07) 0.18 0.84 (0.63–1.12) 0.24
CD14
Rs2569190
 GG 236 194
 GA 360 339 1.15 (0.90–1.46) 0.27 1.18 (0.92–1.51) 0.19 1.27 (0.91–1.78) 0.16
 AA 170 157 1.12 (0.84–1.50) 0.43 1.20 (0.89–1.61) 0.24 1.46 (0.98–2.19) 0.06
 GA or AA 530 496 1.14 (0.91–1.43) 0.26 1.18 (0.94–1.50) 0.15 1.32 (0.96–1.82) 0.08
TIRAP
rs8177374
 CC 556 521
 CT 185 159 0.92 (0.72–1.17) 0.49 0.99 (0.77–1.27) 0.94 1.38 (0.99–1.91) 0.06
 TT 21 15 0.76 (0.39–1.49) 0.43 0.76 (0.38–1.53) 0.45 1.31 (0.55–3.12) 0.55
 CT or TT 206 174 0.90 (0.71–1.14) 0.39 0.97 (0.76–1.23) 0.81 1.38 (1.00–1.89) 0.047
SUMO4
rs237025
 TT 215 195
 TC 362 358 1.09 (0.86–1.39) 0.48 1.08 (0.84–1.38) 0.55 1.04 (0.75–1.44) 0.80
 CC 195 136 0.77 (0.57–1.03) 0.08 0.75 (0.55–1.01) 0.06 0.55 (0.36–0.84) 0.01
 TC or CC 557 494 0.98 (0.78–1.23) 0.85 0.96 (0.76–1.22) 0.75 0.87 (0.64–1.19) 0.38
NFKBIA
rs696
 GG 298 259
 GA 366 336 1.06 (0.85–1.32) 0.63 1.06 (0.84–1.33) 0.64 1.02 (0.75–1.39) 0.88
 AA 101 90 1.03 (0.74–1.43) 0.88 0.97 (0.69–1.36) 0.86 1.07 (0.67–1.69) 0.78
 GA or AA 467 426 1.05 (0.85–1.30) 0.65 1.04 (0.84–1.29) 0.73 1.03 (0.77–1.38) 0.84
NFKB1
rs28362491
 Ins/Ins 269 258
 Ins/− 376 316 0.88 (0.70–1.10) 0.25 0.89 (0.70–1.12) 0.31 0.74 (0.54–1.01) 0.06
 −/− 122 100 0.85 (0.62–1.17) 0.33 0.82 (0.59–1.13) 0.22 0.78 (0.51–1.19) 0.25
 Ins/− or −/− 498 416 0.87 (0.70–1.08) 0.21 0.87 (0.70–1.08) 0.21 0.75 (0.56–1.01) 0.06
TNF
rs1800629
 GG 527 549
 GA 223 129 0.56 (0.43–0.71) 0.0000032 0.58 (0.45–0.75) 0.000029 0.63 (0.45–0.89) 0.01
 AA 25 9 0.35 (0.16–0.75) 0.01 0.39 (0.18–0.85) 0.02 0.19 (0.04–0.79) 0.02
 GA or AA 248 138 0.53 (0.42–0.68) 0.00000030 0.56 (0.44–0.72) 0.0000047 0.59 (0.42–0.82) 0.0018
TNF
rs361525
 GG 708 669
 GA 60 30 0.53 (0.34–0.83) 0.01 0.52 (0.32–0.82) 0.0049 0.61 (0.33–1.12) 0.11
 AA 3 0 1.00 (1.00–1.00) 1.00 1.00 (1.00–1.00) 1.00 1.00 (1.00–1.00) 1.00
 GA or AA 63 30 0.50 (0.32–0.79) 0.0027 0.49 (0.31–0.78) 0.0024 0.58 (0.32–1.05) 0.07
TNFRSF1A
rs4149570
 GG 307 217
 GT 355 339 1.35 (1.07–1.70) 0.01 1.33 (1.05–1.68) 0.02 1.46 (1.06–2.00) 0.02
 TT 109 132 1.71 (1.26–2.33) 0.00060 1.79 (1.31–2.46) 0.00027 2.26 (1.48–3.47) 0.00017
 GT or TT 464 471 1.44 (1.16–1.78) 0.0010 1.44 (1.15–1.80) 0.0013 1.64 (1.21–2.22) 0.0014
TNFAIP3
rs6927172
 CC 473 415
 CG 264 245 1.06 (0.85–1.32) 0.61 1.06 (0.85–1.33) 0.61 1.03 (0.76–1.39) 0.85
 GG 40 25 0.71 (0.42–1.19) 0.20 0.70 (0.41–1.19) 0.18 0.51 (0.23–1.10) 0.09
 CG or GG 304 270 1.01 (0.82–1.25) 0.91 1.01 (0.82–1.26) 0.91 0.95 (0.71–1.27) 0.73
TGFB1
rs1800469
 CC 383 344
 CT 297 299 1.12 (0.90–1.39) 0.30 1.08 (0.87–1.35) 0.48 1.28 (0.95–1.71) 0.11
 TT 86 53 0.69 (0.47–1.00) 0.047 0.69 (0.47–1.02) 0.06 0.69 (0.40–1.17) 0.17
 CT or TT 383 352 1.02 (0.83–1.26) 0.83 1.00 (0.81–1.23) 0.97 1.14 (0.86–1.52) 0.35
PTPN22
rs2476601
 GG 588 557
 GA 166 122 0.78 (0.60–1.01) 0.06 0.77 (0.59–1.00) 0.05 0.75 (0.52–1.09) 0.13
 AA 11 6 0.58 (0.21–1.57) 0.28 0.57 (0.20–1.58) 0.28 0.83 (0.21–3.28) 0.80
 GA or AA 177 128 0.76 (0.59–0.99) 0.04 0.76 (0.58–0.98) 0.04 0.76 (0.53–1.09) 0.13
PPARG
rs1801282
 CC 548 511
 CG 207 167 0.87 (0.68–1.10) 0.23 0.85 (0.66–1.08) 0.18 0.87 (0.63–1.21) 0.42
 GG 14 15 1.15 (0.55–2.40) 0.71 1.33 (0.62–2.83) 0.46 1.54 (0.60–3.98) 0.37
 CG or GG 221 182 0.88 (0.70–1.11) 0.29 0.88 (0.69–1.11) 0.27 0.91 (0.67–1.26) 0.58
IL1B
rs4848306
 GG 246 215
 GA 373 352 1.08 (0.85–1.36) 0.52 1.09 (0.86–1.39) 0.48 1.16 (0.84–1.60) 0.38
 AA 151 125 0.95 (0.70–1.28) 0.72 0.96 (0.71–1.31) 0.81 0.88 (0.57–1.34) 0.55
 GA or AA 524 477 1.04 (0.83–1.30) 0.72 1.06 (0.84–1.33) 0.64 1.08 (0.79–1.46) 0.64
IL1B
rs1143623
 GG 401 365
 GC 316 278 0.97 (0.78–1.20) 0.76 0.98 (0.79–1.22) 0.87 1.07 (0.80–1.44) 0.66
 CC 55 52 1.04 (0.69–1.56) 0.85 1.12 (0.74–1.69) 0.59 0.87 (0.48–1.57) 0.64
 GC or CC 371 330 0.98 (0.80–1.20) 0.83 1.00 (0.81–1.24) 0.98 1.04 (0.78–1.38) 0.79
IL1B
rs1143627
 TT 340 305
 TC 339 305 1.00 (0.81–1.25) 0.98 1.00 (0.79–1.25) 0.97 1.05 (0.78–1.42) 0.75
 CC 97 86 0.99 (0.71–1.37) 0.94 1.01 (0.72–1.41) 0.95 0.85 (0.53–1.36) 0.50
 TC or CC 436 391 1.00 (0.81–1.23) 1.00 1.00 (0.81–1.24) 1.00 1.00 (0.76–1.34) 0.97
IL1RN
rs4251961
 TT 298 247
 TC 360 324 1.09 (0.87–1.36) 0.47 1.04 (0.83–1.32) 0.71 1.22 (0.89–1.67) 0.21
 CC 112 105 1.13 (0.83–1.55) 0.44 1.05 (0.76–1.46) 0.76 1.41 (0.92–2.17) 0.12
 TC or CC 472 429 1.10 (0.89–1.36) 0.40 1.05 (0.84–1.30) 0.68 1.26 (0.94–1.71) 0.12
IL4R
rs1805010
 AA 209 201
 AG 410 317 0.80 (0.63–1.02) 0.08 0.79 (0.62–1.02) 0.07 0.73 (0.52–1.02) 0.07
 GG 157 133 0.88 (0.65–1.19) 0.41 0.91 (0.67–1.24) 0.55 0.87 (0.58–1.33) 0.53
 AG or GG 567 450 0.83 (0.66–1.04) 0.10 0.83 (0.65–1.05) 0.12 0.77 (0.56–1.06) 0.11
IL6
rs10499563
 TT 476 439
 TC 259 225 0.94 (0.76–1.17) 0.60 0.94 (0.75–1.18) 0.60 0.77 (0.57–1.05) 0.10
 CC 35 26 0.81 (0.48–1.36) 0.42 0.72 (0.42–1.25) 0.24 0.80 (0.39–1.63) 0.53
 TC or CC 294 251 0.93 (0.75–1.14) 0.48 0.92 (0.74–1.14) 0.43 0.77 (0.57–1.04) 0.09
IL6R
rs4537545
 CC 289 247
 CT 369 324 1.03 (0.82–1.29) 0.82 1.05 (0.83–1.32) 0.71 1.07 (0.79–1.47) 0.65
 TT 117 113 1.13 (0.83–1.54) 0.44 1.18 (0.86–1.63) 0.30 1.17 (0.76–1.79) 0.48
 CT or TT 486 437 1.05 (0.85–1.30) 0.64 1.08 (0.86–1.34) 0.51 1.09 (0.81–1.47) 0.55
IL10
rs1800872
 CC 482 408
 CA 258 225 1.03 (0.83–1.29) 0.79 1.01 (0.80–1.27) 0.94 0.93 (0.68–1.26) 0.63
 AA 35 42 1.42 (0.89–2.26) 0.14 1.35 (0.83–2.18) 0.22 1.47 (0.79–2.73) 0.22
 CA or AA 293 267 1.08 (0.87–1.33) 0.50 1.05 (0.84–1.30) 0.67 0.99 (0.74–1.33) 0.95
IL10
rs3024505
 CC 518 467
 CT 221 200 1.00 (0.80–1.26) 0.97 1.01 (0.80–1.28) 0.95 1.19 (0.87–1.61) 0.28
 TT 22 24 1.21 (0.67–2.19) 0.53 1.32 (0.72–2.42) 0.37 1.80 (0.79–4.12) 0.16
 CT or TT 243 224 1.02 (0.82–1.27) 0.84 1.04 (0.83–1.30) 0.76 1.23 (0.92–1.66) 0.17
IL12B
rs3212217
 GG 499 460
 GC 235 200 0.92 (0.74–1.16) 0.49 0.95 (0.75–1.19) 0.64 0.94 (0.69–1.29) 0.72
 CC 25 21 0.91 (0.50–1.65) 0.76 0.94 (0.51–1.72) 0.84 0.57 (0.23–1.41) 0.22
 GC or CC 260 221 0.92 (0.74–1.15) 0.47 0.95 (0.76–1.19) 0.63 0.91 (0.67–1.23) 0.53
IL12B
rs6887695
 GG 385 324
 GC 293 301 1.22 (0.98–1.52) 0.07 1.24 (0.99–1.55) 0.06 1.31 (0.97–1.77) 0.07
 CC 72 70 1.16 (0.81–1.66) 0.43 1.16 (0.80–1.69) 0.43 0.98 (0.59–1.61) 0.94
 GC or CC 365 371 1.21 (0.98–1.49) 0.07 1.22 (0.99–1.51) 0.06 1.24 (0.93–1.64) 0.14
IL12RB1
rs401502
 CC 360 304
 CG 303 311 1.22 (0.98–1.51) 0.08 1.21 (0.96–1.51) 0.10 1.19 (0.88–1.61) 0.26
 GG 87 70 0.95 (0.67–1.35) 0.79 0.97 (0.68–1.39) 0.87 1.18 (0.74–1.88) 0.48
 CG or GG 390 381 1.16 (0.94–1.42) 0.17 1.15 (0.93–1.43) 0.19 1.19 (0.89–1.58) 0.24
IL17A
rs2275913
 GG 340 307
 GA 336 301 0.99 (0.80–1.24) 0.94 0.98 (0.79–1.23) 0.89 0.90 (0.67–1.22) 0.51
 AA 95 84 0.98 (0.70–1.36) 0.90 1.00 (0.71–1.40) 0.98 1.00 (0.63–1.57) 0.99
 GA or AA 431 385 0.99 (0.80–1.22) 0.92 0.99 (0.80–1.22) 0.89 0.92 (0.69–1.22) 0.57
IL18
rs187238
 GG 387 380
 GC 312 259 0.85 (0.68–1.05) 0.13 0.83 (0.66–1.03) 0.09 0.74 (0.55–1.00) 0.049
 CC 64 41 0.65 (0.43–0.99) 0.04 0.69 (0.45–1.06) 0.09 0.58 (0.32–1.04) 0.07
 GC or CC 376 300 0.81 (0.66–1.00) 0.0499 0.80 (0.65–0.99) 0.04 0.71 (0.53–0.95) 0.02
IL18
rs1946518
 GG 282 259
 GT 363 329 0.99 (0.79–1.24) 0.91 0.96 (0.76–1.21) 0.71 0.89 (0.65–1.21) 0.45
 TT 113 97 0.93 (0.68–1.29) 0.68 0.95 (0.68–1.31) 0.74 0.80 (0.51–1.24) 0.32
 GT or TT 476 426 0.97 (0.79–1.21) 0.81 0.96 (0.77–1.19) 0.68 0.86 (0.64–1.16) 0.32
IL23R
rs11209026
 GG 680 646
 GA 89 50 0.59 (0.41–0.85) 0.0045 0.63 (0.43–0.91) 0.02 0.64 (0.38–1.05) 0.08
 AA 5 1 1.00 (1.00–1.00) 1.00 1.00 (1.00–1.00) 1.00 1.00 (1.00–1.00) 1.00
 GA or AA 94 51 0.57 (0.40–0.82) 0.0021 0.60 (0.42–0.87) 0.01 0.63 (0.38–1.03) 0.06
IFNG
rs2430561
 TT 199 181
 TA 398 369 1.02 (0.80–1.30) 0.88 1.01 (0.79–1.30) 0.92 1.08 (0.77–1.52) 0.65
 AA 161 139 0.95 (0.70–1.29) 0.74 0.97 (0.71–1.32) 0.85 1.09 (0.72–1.64) 0.68
 TA or AA 559 508 1.00 (0.79–1.26) 0.99 1.00 (0.79–1.27) 0.99 1.08 (0.79–1.50) 0.62
IFNGR1
rs2234711
 TT 290 232
 TC 361 348 1.20 (0.96–1.51) 0.11 1.20 (0.95–1.51) 0.12 1.15 (0.84–1.57) 0.40
 CC 119 108 1.13 (0.83–1.55) 0.43 1.09 (0.79–1.50) 0.60 1.11 (0.72–1.70) 0.65
 TC or CC 480 456 1.19 (0.96–1.47) 0.12 1.17 (0.94–1.46) 0.16 1.14 (0.84–1.53) 0.40
IFNGR2
rs8126756
 TT 553 522
 TC 168 130 0.82 (0.63–1.06) 0.13 0.83 (0.64–1.09) 0.18 0.86 (0.60–1.24) 0.42
 CC 18 12 0.71 (0.34–1.48) 0.36 0.69 (0.32–1.49) 0.35 0.53 (0.18–1.54) 0.24
 TC or CC 186 142 0.81 (0.63–1.04) 0.09 0.82 (0.64–1.06) 0.13 0.83 (0.59–1.17) 0.28
IFNGR2
rs17882748
 CC 199 173
 CT 391 341 1.00 (0.78–1.29) 0.98 1.00 (0.77–1.30) 0.99 1.01 (0.71–1.42) 0.97
 TT 153 174 1.31 (0.97–1.76) 0.08 1.31 (0.97–1.78) 0.08 1.16 (0.77–1.73) 0.48
 CT or TT 544 515 1.09 (0.86–1.38) 0.48 1.09 (0.86–1.39) 0.48 1.05 (0.76–1.45) 0.76
TBX21
rs17250932
 TT 526 497
 TC 210 179 0.90 (0.71–1.14) 0.39 0.94 (0.74–1.19) 0.61 0.84 (0.60–1.17) 0.30
 CC 32 19 0.63 (0.35–1.12) 0.12 0.66 (0.36–1.19) 0.17 0.37 (0.14–0.98) 0.046
 TC or CC 242 198 0.87 (0.69–1.08) 0.21 0.90 (0.72–1.14) 0.39 0.78 (0.56–1.07) 0.12
NLRP1
rs2670660
 AA 222 202
 AG 390 328 0.92 (0.73–1.18) 0.52 0.96 (0.75–1.23) 0.73 1.12 (0.80–1.56) 0.52
 GG 154 154 1.10 (0.82–1.47) 0.53 1.11 (0.82–1.49) 0.51 1.12 (0.75–1.67) 0.59
 AG or GG 544 482 0.97 (0.78–1.22) 0.82 1.00 (0.79–1.26) 0.98 1.11 (0.81–1.52) 0.50
NLRP1
rs878329
 GG 217 206
 GC 394 333 0.89 (0.70–1.13) 0.34 0.89 (0.69–1.14) 0.35 0.99 (0.71–1.38) 0.93
 CC 155 155 1.05 (0.79–1.41) 0.73 1.05 (0.78–1.41) 0.75 1.03 (0.69–1.54) 0.90
 GC or CC 549 488 0.94 (0.75–1.17) 0.57 0.93 (0.74–1.18) 0.56 1.00 (0.73–1.36) 0.98
NLRP3
rs10754558
 CC 294 248
 CG 355 324 1.08 (0.86–1.36) 0.50 1.06 (0.84–1.34) 0.61 1.10 (0.81–1.51) 0.54
 GG 111 116 1.24 (0.91–1.69) 0.18 1.25 (0.91–1.71) 0.17 1.11 (0.71–1.72) 0.65
 CG or GG 466 440 1.12 (0.90–1.39) 0.30 1.11 (0.89–1.38) 0.36 1.11 (0.82–1.49) 0.51
NLRP3
rs4612666
 CC 435 360
 CT 280 277 1.20 (0.96–1.49) 0.11 1.23 (0.99–1.54) 0.07 1.28 (0.95–1.72) 0.10
 TT 53 48 1.09 (0.72–1.66) 0.67 1.19 (0.78–1.82) 0.41 1.07 (0.59–1.94) 0.82
 CT or TT 333 325 1.18 (0.96–1.45) 0.12 1.23 (0.99–1.52) 0.06 1.24 (0.94–1.65) 0.13
CARD8
rs2043211
 AA 321 298
 AT 342 316 1.00 (0.80–1.24) 0.97 0.98 (0.79–1.23) 0.89 0.90 (0.67–1.22) 0.50
 TT 94 78 0.89 (0.64–1.25) 0.52 0.89 (0.63–1.26) 0.50 0.91 (0.57–1.44) 0.68
 AT or TT 436 394 0.97 (0.79–1.20) 0.80 0.96 (0.78–1.19) 0.72 0.90 (0.67–1.19) 0.45
JAK2
rs12343867
 TT 398 358
 TC 299 263 0.98 (0.79–1.22) 0.84 0.96 (0.76–1.20) 0.69 0.82 (0.61–1.12) 0.21
 CC 61 65 1.18 (0.81–1.73) 0.38 1.11 (0.75–1.63) 0.61 1.03 (0.62–1.71) 0.91
 TC or CC 360 328 1.01 (0.82–1.25) 0.90 0.98 (0.79–1.21) 0.86 0.86 (0.64–1.14) 0.29
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Table 2.

Biological interpretation of the single nucleotide polymorphisms (SNPs) associated with ankylosing spondylitis (AS)

Gene Rs-number Pathway Model OR (95% CI) P-value / Bonferronia Effect of minor-allele Biological interpretation
TLR1 rs4833095 Pathogen recognition CC vs TT 2.59 (1.48–4.51) 0.00081 / 0.04 743C increase TLR1 level in PBMC [56] Increased TLR1 level was associated with increased risk of AS. This could indicate that a genetically determined high activity of the NFkB pathway, and thus high TNF-α and IL-17 activity, was associated with increased risk of AS.
TLR4 rs1554973 Pathogen recognition CC vs TT 0.55 (0.34–0.86) 0.010 / 0.51 Unknown [67] –
LY96 rs11465996 Pathogen recognition GG vs CC 0.68 (0.46–1.00) 0.049 / 1.00 -1625G increase MD-2 and TNF-α levels in human U937 cells and whole blood leukocytes [57] Increased MD-2 and TNF-α level was associated with a reduced risk of AS. In contrast to the other results this indicate that genetically determined high TNF-driven inflammatory response was associated with reduced risk of AS.
TNF rs1800629 Cytokines GA or AA vs GG 0.56 (0.44–0.72) 0.0000047 / 0.00024 -308A increase expression in jurkat cells [65], reduce mRNA level in PBMC and serum [48] or no association was found [49] Reduced TNF-α mRNA level was associated with reduced risk of AS. This could indicate that genetically determined high TNF-driven inflammatory response was associated with increased risk of AS.
TNF rs361525 Cytokines GA or AA vs GG 0.49 (0.31–0.78) 0.0024 / 0.12 -238A reduce expression in PBMC [49] Reduced TNF-α expression was associated with reduced risk of AS. This indicates that genetically determined high TNF-driven inflammatory response was associated with increased risk of AS.
TNFRSF1A rs4149570 Cytokines GT or TT vs GG 1.44 (1.15–1.80) 0.0013 / 0.066b -609 T increase expression in PBMC [50] Increased TNF-α receptor 1 expression was associated with increased risk of AS. This indicates that genetically determined high TNF-driven inflammatory response was associated with increased risk of AS.
PTPN22 rs2476601 Immune response GA or AA vs GG 0.76 (0.58–0.98) 0.037 / 1.00 1858A reduce TNF-α level in serum [51] Reduced TNF-α level was associated with reduced risk of AS. This indicates that genetically determined high TNF-driven inflammatory response was associated with increased risk of AS.
IL18 rs187238 Cytokines GC or CC vs GG 0.80 (0.65–0.99) 0.044 / 1.00 -137C reduce IL-18 level in serum [53] and expression in PBMC [54] Reduced IL-18 expression, and thus reduced IL-17 and TNF-α activity, was associated with reduced risk of AS.
This indicates that a genetically determined high activity of the IL23/IL17 pathway was associated with increased risk of AS.
IL23R rs11209026 Cytokines GA or AA vs GG 0.60 (0.42–0.87) 0.0071 / 0.36 rs11209026A reduce IL-17 level in PBMC [52] Reduced IL-17 level was associated with reduced risk of AS. This indicates that a genetically determined high activity of the IL23/IL17 pathway was associated with increased risk of AS.
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OR Odds ratio

95% CI 95% confidence interval

PBMC peripheral blood mononuclear cell

aThe Bonferroni calculations were based on the 51 SNPs assessed in this study

bThe TNFRSF1A (rs4149570) TT vs GG: OR: 1.79, 95% CI: 1.31–2.41, p = 0.00027, Bonferroni = 0.014

DNA extraction (Maxwell 16 LEV Blood DNA Kit; Promega, Madison, WI, USA) was performed as described by Bank et al. [10]. For the healthy controls, DNA was extracted from EDTA-stabilized peripheral blood by either PureGene (Qiagen, Hilden, Germany) or Wizard Genomic (Promega, Madison, Wisconsin, USA) DNA purification kit according to the manufacturers` instructions [11–17]. Competitive Allele-Specific Polymerase chain reaction (KASP™), an end-point PCR technology, was used by LGC Genomics for genotyping (LGC Genomics, Hoddesdon, United Kingdom) (http://www.lgcgenomics.com/).

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 used to test for deviation from Hardy-Weinberg equilibrium in the healthy controls and for haplotype analysis (Tables 3, 4, 5 and 6).

Table 3.

Association of the TLR2 haplotype combinations and risk of ankylosing spondylitis (AS). The haplotype combinations in TLR2 described 93% of the genotypes observed

Haplotype combinations Haplotypes NAS (%) NControl (%) ORa (95% CI) P-value
rs4696480 A > T rs11938228 C > A rs3804099 T>Cb
11 T:T A:A T:T 69 (11) 76 (10) 1.00 – –
22 A:A C:C C:C 72 (11) 74 (10) 1.07 0.68–1.70 0.82
33 A:A C:C T:T 28 (4) 34 (5) 0.91 0.50–1.65 0.76
44 T:T C:C C:C 14 (2) 10 (1) 1.52 0.64–3.70 0.38
12 T:A C:A C:T 158 (24) 197 (27) 0.88 0.60–1.30 0.55
13 T:A C:A T:T 76 (12) 103 (14) 0.81 0.52–1.26 0.37
14 T:T C:A C:T 59 (9) 49 (7) 1.33 0.80–2.19 0.31
23 A:A C:C C:T 77 (12) 89 (12) 0.95 0.61–1.49 0.91
24 T:A C:C C:C 52 (8) 55 (8) 1.04 0.63–1.72 0.90
34 T:A C:C C:T 51 (8) 44 (6) 1.28 0.76–2.14 0.43
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OR Odds ratio

aOR was calculated for each haplotype combination by using the haplotype 11 as reference group

bThe variant allele of rs3804099T T > C has been shown to decrease TNF-α, IL-1β & IL-6 level [68]

Table 4.

Association between TLR4 haplotype combinations and risk of ankylosing spondylitis (AS). The haplotype combinations in TLR4 described 94% of the genotypes observed

Haplotype combinations Haplotypes NAS (%) NControl (%) ORa (95% CI) P-value
rs12377632
T > C		 rs1554973
T > C		 rs5030728
G > A
11 C:C T:T G:G 95 (14) 101 (14) 1.00 – –
22 T:T T:T A:A 69 (10) 74 (10) 0.99 0.64–1.53 1.00
33 T:T C:C G:G 29 (4) 57 (8) 0.54 0.32–0.92 0.03
44 T:T T:T G:G 3 (0) 5 (1) 0.64 0.15–2.74 0.72
12 T:C T:T G:A 154 (23) 188 (25) 0.87 0.61–1.24 0.47
13 T:C T:C G:G 126 (19) 129 (17) 1.04 0.72–1.51 0.85
14 T:C T:T G:G 30 (5) 32 (4) 1.00 0.56–1.77 1.00
23 T:T T:C G:A 99 (15) 106 (14) 0.99 0.67–1.47 1.00
24 T:T T:T G:A 31 (5) 24 (3) 1.37 0.75–2.51 0.36
34 T:T T:C G:G 28 (4) 26 (4) 1.14 0.63–2.09 0.76
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OR Odds ratio

The biological effect of the three polymorphisms in TLR4 was unknown

aOR was calculated for each haplotype combination by using the haplotype 11 as reference group

Table 5.

Association between IL1B haplotype combinations and risk of ankylosing spondylitis (AS). The haplotype combinations in IL1B described 97% of the genotypes observed

Haplotype combinations Haplotypes NAS (%) NControl (%) ORa (95% CI) P-value
rs4848306
-3737G > A [69, 70]		 rs1143623
-1464G > C [69, 71]		 rs1143627
-31 T > C [69, 71, 72]
11 A:A G:G T:T 125 (18) 148 (20) 1.00 – –
22 G:G C:C C:C 52 (8) 54 (7) 1.14 0.73–1.79 0.65
33 G:G G:G T:T 32 (5) 41 (5) 0.92 0.55–1.55 0.79
44 G:G G:G C:C 5 (1) 3 (0) 1.97 0.46–8.42 0.48
12 A:G G:C T:C 163 (24) 185 (24) 1.04 0.76–1.43 0.81
13 A:G G:G T:T 141 (20) 147 (19) 1.14 0.82–1.58 0.50
14 A:G G:G T:C 44 (6) 38 (5) 1.37 0.84–2.25 0.26
23 G:G C:G C:T 84 (12) 92 (12) 1.08 0.74–1.58 0.70
24 G:G C:G C:C 28 (4) 34 (4) 0.98 0.56–1.70 1.00
34 G:G G:G T:C 14 (2) 16 (2) 1.04 0.49–2.21 1.00
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OR Odds ratio

The variant allele of −3737 G > A [69], −1464 G > C [70] and − 31 T > C [71, 72] have been shown to decrease IL-1β level [69–72]

aOR was calculated for each haplotype combination by using the haplotype 11 as reference group

Table 6.

Association of the TNF haplotype combinations and risk of ankylosing spondylitis (AS). The haplotype combinations in TNF described 97% of the genotypes observed

Haplotype combinations Haplotypes NAS (%) NControl (%) ORa (95% CI) P-value
rs361525 G>Ab rs1800629 G>Ac
11 G:G G:G 523 (76) 469 (61) 1.00 – –
22 G:G A:A 9 (1) 25 (3) 0.32 (0.15–0.70) 0.005
12 G:G G:A 125 (18) 210 (28) 0.53 (0.41–0.69) < 0.0001
13 G:A G:G 26 (4) 47 (6) 0.50 (0.30–0.81) 0.007
14 G:A G:A 4 (1) 12 (2) 0.30 (0.10–0.93) 0.05
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OR Odds ratio

aOR was calculated for each haplotype combination by using the haplotype 11 as reference group

bThe variant allele of TNF -238A rs361525A G > A has been shown to reduce expression of TNF-α [49]

cThe variant allele of TNF -308A rs1800629 G > A has been shown to reduce mRNA level [48]

Statistical analyses were performed using STATA version 15 (StataCorp LP, College Station, TX, USA).

Results

Study population

Among the patients with AS the median age was 32 years (SD: 11.5) and 68% (483/709) were males. The healthy controls had a median age of 43 years (SD: 11.5) and 52% (411/384) were males. Among the patients 37% (118/323), 23% (73/323), and 41% (132/323) and among the controls 26% (207/788), 24% (189/788), and 50% (392/788) were current smokers, former smokers and never smokers, respectively. HLA-B27 staus was available for 498 patients of which 83% (411/498) were positive. Sixty percent (427/709) of the patients were treated with anti-TNF.

The genotype distributions among the healthy controls deviated from Hardy-Weinberg equilibrium for TLR1 (743 T > C (rs4833095)) (p = 0.03), TLR2 (− 16,934 A > T (rs4696480)) (p = 0.02), TLR4 (rs1554973 T > C) (p = 0.03), TLR9 (1174 G > A (rs352139)) (p = 0.02) and TGFB1 (− 509 C > T (rs1800469)) (p = 0.02). After correction for multiple testing, all SNPs studied were in Hardy-Weinberg equilibrium.

Polymorphisms associated with susceptibility of AS

In the age and sex adjusted analysis, the homozygous variant genotype of TLR1 743 T > C (rs4833095) (OR: 2.59, 95% CI: 1.48–4.51, p = 0.0008) and the combined homozygous and the heterozygous variant genotypes of TNFRSF1A -609 G > T (rs4149570) (OR: 1.44, 95% CI: 1.15–1.80, p = 0.001) were associated with increased risk of AS. The homozygous variant genotype of TLR4 T > C (rs1554973) (OR: 0.55, 95% CI: 0.34–0.86, p = 0.01) and LY96–1625 C > G (rs11465996) (OR: 0.68, 95% CI: 0.46–1.00, p = 0.05), and the combined homozygous and the heterozygous variant genotypes of TNF -308 G > A (rs1800629) (OR: 0.56, 95% CI: 0.44–0.72, p = 0.000005), TNF -238 G > A (rs361525) (OR: 0.49, 95% CI: 0.31–0.78, p = 0.002), PTPN22 1858 G > A (rs2476601) (OR: 0.76, 95% CI: 0.58–0.98, p = 0.04), IL18–137 G > C (rs187238) (OR: 0.80, 95% CI: 0.65–0.99, p = 0.04), and IL23R G > A (rs11209026) (OR: 0.60, 95% CI: 0.42–0.87, p = 0.01) were associated with reduced risk of AS (Table 1).

After Bonferroni correction for multiple testing the homozygous variant genotype of TLR1 743 T > C (rs4833095) (OR: 2.59, 95% 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 the 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 (Table 2).

SNPs associated with AS and the biological effect of the SNPs are summarized in Table 2.

Haplotype analysis

Haplotype analyses of TLR2, TLR4, IL1B and TNF are shown in Tables 3, 4, 5 and 6, respectively.

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.

Discussion

In this case-control study, polymorphisms in a: the TNF-α (TNF (rs1800629 and rs361525), TNFRSF1A (rs4149570), and PTPN22 (rs2476601)), b: the IL23/IL17 (IL23R (rs11209026), and IL18 (rs187238)), or c: the NFkB (TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996)) pathways were associated with risk of AS.

The found assocaitions for TNF (rs1800629) [19–22], TNFRSF1A (rs4149570) [23], and IL23R (rs11209026) [24–33] are in agreement with other case-control studies. Furthermore, Zhao et al. found that the variant allele of NLRP3 (rs4612666) was associated with increased risk of AS in Chinese patients [23]. In our study we found a trend for associations of the variant allele of NLRP3 (rs4612666) with increased risk of AS (p = 0.06). However, our results are in contrast to a meta-analysis of the PTPN22 (rs2476601) polymorphism that did not find an association with AS [34]. Finally, we identified novel risk loci in TNF (rs361525), IL18 (rs187238), TLR1 (rs4833095), TLR4 (rs1554973), and LY96 (rs11465996) that need validation in independent cohorts.

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 [35–47]. 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.

Both TNF-α [58] and interleukin-17 inhibitors [59] have been shown to reduce inflammation and improve symptoms in patients with AS [60]. Furthermore, increased levels of TNF-α, IL-17, IL-23, IL-1β, and IL-6 have been found in sera and synovial fluid from AS patients [61–64]. The genetic associations between AS and the polymorphisms in TLR1, TLR4, LY96, TNF, TNFRSF1A, IL18, and IL23R found in this study, could potentially – in part – explain this altered cytokine milieu present in AS patients.

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 (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 the risk of AS.

Acknowledgments

We thank Ewa Kogutowska and Mette Errebo Rønne, Statens Serum Institut, for laboratory support; and Niels Steen Krogh, Zitelab Aps, Copenhagen, Denmark for database management. We also thank Department of Medicine, Viborg Regional Hospital, Denmark and OPEN (Odense Patient data Explorative Network), Odense University Hospital, Denmark for supporting this work.

In memory of Niels Henrik Heegaard:

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.

Funding

This study was funded by the Danish Rheumatism Association (A1923, A3037, and A3570 - www. Gigtforeningen.dk) and Region of Southern Denmark’s PhD Fund, 12/7725 (www. Regionsyddanmark.dk).

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.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Footnotes

Niels Henrik Heegaard is deceased. This paper is dedicated to his memory.

Contributor Information

Jacob Sode, Email: Jacob.Sode@gmail.com.

Steffen Bank, Email: stb@mb.au.dk.

Ulla Vogel, Email: UBV@arbejdsmiljoforskning.dk.

Paal Skytt Andersen, Email: PSA@SSI.DK.

Signe Bek Sørensen, Email: Signe.Bek.Sorensen@rsyd.dk.

Anders Bo Bojesen, Email: anders@socioskop.dk.

Malene Rohr Andersen, Email: Malene.Rohr.Andersen@regionh.dk.

Ivan Brandslund, Email: ivan.brandslund@rsyd.dk.

Ram Benny Dessau, Email: ramd@regionsjaelland.dk.

Hans Jürgen Hoffmann, Email: hans.jurgen.hoffmann@ki.au.dk.

Bente Glintborg, Email: glintborg@dadlnet.dk.

Merete Lund Hetland, Email: merete.hetland@dadlnet.dk.

Henning Locht, Email: locht@dadlnet.dk.

Vibeke Andersen, Email: Vibeke.Andersen1@rsyd.dk.

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Associated Data

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Data Availability Statement

The datasets used during the current study are available from the corresponding author on reasonable request.

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