Introduction
The immediate cause of gout is the deposition of monosodium urate (MSU) crystals in and around body tissues, particularly joints [
1]. Initially, these deposits trigger a localised and self-limiting inflammatory response (acute gouty arthritis), which becomes increasingly frequent and severe, involving multiple joints and associated with fever. Monosodium urate crystals form under hyperuricaemic conditions when serum urate levels exceed the physiological saturation level (approximately 6.8 mg/dL; approximately 0.41 mM). The most significant biological cause of hyperuricaemia is relatively low renal clearance of uric acid [
2,
3]. This is consistent with findings from genome-wide association studies in which 28 loci associated with serum urate levels have been identified, some of which are in genes involved in renal uric acid handling [
4,
5]. Predictably most, but not all, of the 28 loci have been associated with gout [
4,
6].
Although hyperuricaemia is a prerequisite for MSU formation, only a relatively small proportion of individuals with hyperuricaemia develop gout [
7]. This indicates that beside genetic variants associated with urate metabolism and excretion, other factors contribute to the pathogenesis of gout. MSU crystals play an important role in activation of the innate immune system [
8] and the recognition of gout as an auto-inflammatory disorder is consistent with the results of functional studies [
9,
10]. Variations within genes of the innate immune system may therefore determine whether MSU crystals trigger an inflammatory reaction in susceptible individuals, leading to acute gout; while in others, no inflammation is elicited. Genetic variants that influence the activation and function of the NOD-like Receptor Pyrin containing 3 (NLRP3) inflammasome are candidate genes in this context [
11]. The multi-protein inflammasome complex, comprising the NLRP3 polypeptide, ASC or PYCARD (apoptosis-associated speck-like protein containing a CARD) and caspase-1 [
12] forms when monocytes and macrophages encounter damaged and pathogen-associated molecular pattern proteins (DAMPs and PAMP; e.g., bacterial lipopolysaccharide or MSU crystals) and leads to activation of caspase-1. Active caspase-1 processes the pro-interleukin (IL)-1β to the mature pro-inflammatory cytokine IL-1β that is then secreted [
12].
CARD8 (also known as TUCAN or Cardinal) is a protein with a caspase-domain that interacts with caspase-1 and inhibits its activation [
13] and also with a FIIND domain that binds to NLRP3 preventing its recruitment into the active inflammasome complex [
14,
15]. Genetic associations between variants of CARD8 and autoimmune diseases have been previously reported (reviewed in [
16]). The T allele of
CARD8 rs2043211 (C10X) has been associated with increased risk of gout in Chinese [
17], and
rs2149356 in toll-like receptor 4 (TLR4), a receptor functionally implicated in MSU-stimulated inflammation [
18], has also been associated with serum IL-1β levels and the risk of gout in Han Chinese [
19]. However, using a haplotype tagging approach, there is no evidence of association between
NLRP3 and gout in Chinese [
20].
1
Our aim was to extend the findings from the Han Chinese population [
17] and to test genetic variants influencing inflammasome function for association with gout in other population groups. Eleven functional variants were tested in eight genes involved in the MSU crystal-mediated activation of the NLRP3-inflammasome and production of mature IL-1β for association with gout in people of European and New Zealand (NZ) Polynesian (Māori and Pacific Island) ancestry. The prevalence of gout in the NZ Polynesian population is 6–8 % (compared to 3 % in NZ European), exhibiting the highest prevalence worldwide [
21,
22].
Results
Eleven functional genetic variants in
NLRP3, CARD8, IL1B, DAPK1, TXNIP, TLR2, P2XR7, MYD88 and
CD14 were selected from the literature (Table
2) and genotyped; genotype distributions are presented in Additional file
2. There was nominal allelic association (
P <0.05) for three variants in the combined European and Polynesian analysis (Table
3) -
IL1B, CARD8 and
CD14 (OR = 1.10, 1.12 and 1.08, respectively).
CARD8 rs2043211 was also associated with gout in Europeans (OR = 1.11).
Table 2
Eleven genetic variants in NLRP3, CARD8, DAPK1, TXNIP, TLR2, P2XR7, MYD88 and CD14 were selected from the literature
rs10754558 (NLRP3) NLRP3 is a component of the NALP3 inflammasome. | Variant influences transcription (G > C) [ 46] | None |
rs35829419 (NLRP3) | Gain-of-function, with CARD8pC10X identified in arthritic patient with abnormally high IL-1 [ 47, 48] | Minor allele protective against celiac disease in a small study [ 49] |
rs7512998 (NLRP3) | None | |
rs2043211 (CARD8). CARD8 is a negative regulator of IL-1β secretion | C10X encodes a truncated protein that does not abrogate NFkB transcription [ 50]. Can be evaded by alternative splicing [ 51]. Identified as F102I in dbSNP | Interacts with NLRP3 rs35892419 in risk of Crohn’s disease [ 31]. Associated with disease severity in RA [ 50] |
rs1143623 (IL1B). IL-1β is a pro-inflammatory cytokine produced by activated NALP3 inflammasome | Promoter variant influences IL6 levels after fatty-acid rich meals [ 38] and IL-1β levels in small Crohn’s disease study [ 52] | G allele associated with protection from RA [ 53] |
rs4696480 (TLR2). Toll-like receptor involved in NALP3 inflammasome activation | Within binding site for the THP-1-derived nuclear protein, influences reporter expression [ 54] | None |
rs2569190 (CD14). CD14 is an adaptor molecule used by TLR2 |
Rs2569190 T allele alters transcriptional activity [ 42, 43] | On meta-analysis is associated with Crohn’s disease [ 55] and asthma [ 56] |
rs6853 (MYD88). MYD88 is a transducer in the TLR signalling pathway | A allele creates potential miR-562b binding site [ 57] | None |
rs17525809 (P2RX7). Purine receptor involved in a pathway of NALP3 activation via amyloid A | Missense variant (Val > Ala), T allele reduces activity [ 58] | None |
rs4878104 (DAPK1). DAPK1 (death associated kinase) is involved in NALP3 assembly. | Exhibits allelic specific differences in expression [ 59] | None |
rs7212 (TXNIP). TXNIP is thioredoxin-interacting protein required for full inflammasome activation | Increased mRNA expression in smooth muscle cells with G allele [ 60] | None |
Table 3
Analysis of associations between the minor alleles of the eleven variants and gout
NLRP3, rs10754558, G | 0.408 | 0.418 | 0.95 (0.87–1.04) | 0.25 | 0.459 | 0.434 | 1.07 (0.91–1.24) | 0.42 | 0.96 (0.036) | 0.44 |
NLRP3, rs35829419, A | 0.046 | 0.047 | 0.88 (0.71–1.07) | 0.20 | 0.004 | 0.012 | 0.58 (0.20–1.53) | 0.29 | 0.87 (0.098) | 0.15 |
NLRP3, rs7512998, C | 0.170 | 0.160 | 1.09 (0.94–1.27) | 0.25 | 0.033 | 0.045 | 1.03 (0.67–1.60) | 0.88 | 1.08 (0.075) | 0.29 |
CARD8, rs2043211, T | 0.338 | 0.321 | 1.11 (1.01–1.22) | 0.023 | 0.499 | 0.439 | 1.15 (0.98–1.35) | 0.078 | 1.12 (0.042) | 0.007 |
IL1B, rs1143623, G | 0.278 | 0.266 | 1.09 (0.99–1.20) | 0.066 | 0.532 | 0.481 | 1.14 (0.98–1.33) | 0.098 | 1.10 (0.042) | 0.020 |
TLR2, rs4696480, T | 0.495 | 0.499 | 1.02 (0.93–1.11) | 0.68 | 0.482 | 0.490 | 0.98 (0.84–1.15) | 0.84 | 1.01 (0.036) | 0.74 |
CD14, rs2569190, A | 0.496 | 0.475 | 1.08 (0.99–1.18) | 0.070 | 0.576 | 0.542 | 1.07 (0.92–1.26) | 0.37 | 1.08 (0.036) | 0.036 |
MYD88, rs6853, G | 0.112 | 0.120 | 0.88 (0.77–1.00) | 0.059 | 0.026 | 0.028 | 1.18 (0.71–1.98) | 0.52 | 0.90 (0.068) | 0.11 |
P2RX7, rs17525809, C | 0.071 | 0.068 | 1.09 (0.92–1.29) | 0.30 | 0.056 | 0.068 | 0.78 (0.56–1.08) | 0.13 | 1.01 (0.080) | 0.87 |
DAPK1, rs4878104, T | 0.358 | 0.353 | 1.02 (0.93–1.11) | 0.72 | 0.701 | 0.622 | 1.13 (0.96–1.34) | 0.14 | 1.05 (0.044) | 0.32 |
TNXIP, rs7212, G | 0.050 | 0.041 | 1.27 (1.04–1.55) | 0.020 | 0.191 | 0.175 | 1.00 (0.82–1.22) | 0.98 | 1.13 (0.071) | 0.091 |
Because of reported interactions between
NLRP3 and
CARD8 in other auto-inflammatory conditions [
30,
31], and the biological interaction between the inflammasome and IL-1β we tested for pairwise multiplicative interaction between
NLRP3/CARD8,
NLRP3/IL1B and
CARD8/IL1B (Table
4), using only
rs10754558 of
NLRP3 owing to the low MAF of
rs35829419 in both Europeans and Polynesians and the low MAF of
rs7512998 in Polynesians. There was evidence for interaction between
CARD8 and
IL1B (
P = 0.005,
P
c = 0.015), driven by amplification of the risk conferred by the
CARD8 rs2043211 T allele in the presence of the
rs1143623 IL1B minor allele homozygous (GG) genotype. Nominally significant interaction between
NLRP3/IL1B (
P
Nominal
= 0.048) was also observed, although this was not significant after adjustment for multiple testing (
P
c = 0.144).
Table 4
Interaction analysis: genotype combinations of CARD8 rs2043211 and NLRP3 rs10754558, CARD8 rs2043211 and IL1B rs1143623, and NLRP3 rs10754558 and IL1B rs1143623
rs10754558- rs2043211 (NLRP3-CARD8) | | | | |
CC/AA | 318 (0.127) | 1,862 (0.155) | 1.00 | |
CC/AT | 376 (0.150) | 1,722 (0.144) | 1.17 (0.96–1.42) | 0.11 |
CC/TT | 133 (0.053) | 476 (0.040) | 1.55 (1.18–2.04) | 0.001 |
CG/AA | 450 (0.180) | 2,606 (0.217) | 0.94 (0.78–1.13) | 0.53 |
CG/AT | 583 (0.233) | 2,565 (0.214) | 1.16 (0.97–1.38) | 0.11 |
CG/TT | 186 (0.074) | 635 (0.053) | 1.45 (1.11–1.81) | 0.006 |
GG/AA | 172 (0.069) | 964 (0.080) | 0.95 (0.75–1.20) | 0.65 |
GG/AT | 206 (0.082) | 905 (0.076) | 1.22 (0.97–1.54) | 0.085 |
GG/TT | 80 (0.032) | 258 (0.022) | 1.71 (1.23–2.39) | 0.002 |
rs1143623-rs2043211 (IL1B-CARD8) | | | | |
CC/AA | 430 (0.174) | 2,821 (0.236) | 1.00 | |
CC/AT | 488 (0.197) | 2,740 (0.229) | 1.13 (0.96–1.32) | 0.16 |
CC/TT | 136 (0.055) | 644 (0.054) | 1.26 (0.99–1.62) | 0.066 |
CG/AA | 390 (0.157) | 2,182 (0.182) | 1.10 (0.92–1.31) | 0.28 |
CG/AT | 495 (0.200) | 1,979 (0.165) | 1.48 (1.25–1.75) | 4.3 × 10−6
|
CG/TT | 162 (0.065) | 575 (0.048) | 1.74 (1.36–2.22) | 8.7 × 10−6
|
GG/AA | 112 (0.045) | 425 (0.036) | 1.33 (1.00–1.77) | 0.049 |
GG/AT | 166 (0.067) | 465 (0.039) | 1.61 (1.25–2.07) | 2.0 × 10−4
|
GG/TT | 100 (0.040) | 147 (0.012) | 3.77 (2.65–5.34) | <1.0 × 10−6
|
rs10754558- rs1143623 (NLRP3-IL1B) | | | | |
CC/CC | 352 (0.141) | 2,147 (0.179) | 1.00 | |
CC/CG | 346 (0.139) | 1,574 (0.131) | 1.32 (1.09–1.60) | 0.005 |
CC/GG | 128 (0.051) | 340 (0.028) | 2.09 (1.57–2.78) | <1.0 × 10−6
|
CG/CC | 497 (0.200) | 2,963 (0.247) | 1.00 (0.84–1.19) | 1.00 |
CG/CG | 529 (0.212) | 2,312 (0.193) | 1.30 (1.09–1.54) | 0.004 |
CG/GG | 184 (0.074) | 524 (0.044) | 1.43 (1.11–1.85) | 0.005 |
GG/CC | 209 (0.084) | 1,099 (0.092) | 1.22 (0.98–1.52) | 0.072 |
GG/CG | 179 (0.072) | 851 (0.071) | 1.14 (0.90–1.44) | 0.272 |
GG/GG | 66 (0.026) | 173 (0.014) | 1.70 (1.17–2.48) | 0.006 |
Discussion
TLR signalling via the NLRP3 inflammasome has been implicated in gout susceptibility and pathology in vivo and in vitro [
10]; for example, MSU uptake and IL-1β production by bone marrow-derived macrophages derived from TLR2, TLR4 or Myd88 knockout mice is significantly reduced, as is neutrophil influx, in response to subcutaneous injection of MSU in whole animals [
18]. To further elucidate the role of the TLR-inflammasome-IL-1β cascade in gout pathogenesis, eleven candidate genetic variants that functionally impact on this pathway (reviewed in [
10]) were tested for association with gout in a sample set of 2,357 cases, adequately powered to detect association with common variants having an effect size of odds ratio 1.4 or greater (Additional file
1). As discussed below, the nominal evidence for association between gout and
CD14, CARD8 and
IL1B, and the multiplicative interaction between
CARD8 and
IL1B in determining the risk of gout
, support the considerable evidence that TLR-mediated activation of the inflammasome and subsequent release of active IL-1β is a central causal pathogenic pathway of gout [
10,
32].
Variants
rs2043211 (
CARD8),
rs1143623 (
IL1B) and
rs2569190 (
CD14), which were associated with gout, are functional variations in genes directly involved in the NLRP3 signaling pathway, and as such are likely to represent genuine disease-susceptibility loci. CARD8 is an adaptor protein that regulates IL-1β secretion by inhibiting NFKβ signaling (required for the expression of pro-IL-1β) and/or interacting with caspase 1 or NLRP3 to inhibit the generation of active IL-1β from inactive pro-IL-1β [
13,
15]. The effect size of
CARD8 SNP
rs2043211 was consistent between the European and NZ Polynesian samples sets (OR = 1.11,
P = 0.023 and OR = 1.15,
P = 0.078, respectively; combined OR = 1.12,
P = 0.007) and the Chinese sample set reported by Chen et al. (OR = 1.19,
P = 0.08) [
17]
. Collectively our results and the study by Chen et al. [
17] do suggest that the association of the minor allele of
rs2043211 with gout is not a false positive one. SNP
rs2043211 encodes a missense protein variation (C10X or F52I depending on transcript, [
33]) with the minor allele increasing the risk of gout. Although the functional effect of this variation has not been specifically evaluated, the SNP is within an expression quantitative trait locus peak and carriage of the minor allele is associated with decreased CARD8 expression [
34]. It has been inconsistently associated with other auto-inflammatory phenotypes, with some evidence for epistatic interaction with
NLRP3 rs35829419 in determining risk of inflammatory bowel disease and abdominal aortic aneurysms [
31,
35]. However, we found no evidence of interaction between
NLRP3 and
CARD8 in determining risk of gout (Table
4) (although we did not specifically analyse
rs35829419), and it has also been suggested that under certain conditions, ASC-dependent IL1-β production in response to MSU stimulation can occur in the absence of NLRP3 [
36].
IL1B SNP
rs1143623 is within a promoter GATA transcription factor family binding site. Although the minor allele exhibits enhanced protein-binding [
37] and decreased expression in vitro and in vivo, the effect of this variation seems to be influenced both by the wider promoter haplotype [
38‐
40], and the identity of the stimulatory signal; the minor allele shows increased rather than decreased expression in vitro in response to TNF-α [
40], and has also been associated with increased post-prandial triglyceride and IL6 (an effector of IL-1β) levels [
41]. The minor allele is over-represented in gout cases compared to controls (OR = 1.10,
P = 0.020). If this is replicated it would be consistent with an etiological role for increased
IL1B expression in gout. There was evidence of multiplicative interaction with
CARD8 rs2043211 in which the
IL1B rs1143623 minor (risk) allele homozygous genotype appeared to amplify the effect of the minor allele of
rs2043211. This would be consistent with a synergy of greater inflammasome activity (resulting from reduced CARD8) combined with higher levels of pre-IL-1β expression leading to increased production of mature IL-1β in gout.
The final variation nominally associated with gout was
CD14 SNP
rs2569190. This SNP is in the 5’UTR of one of the two CD14 splice variants, with the minor allele (that increases risk of gout) increasing expression in monocytes by decreasing affinity for the inhibitory Sp3 transcription factor [
42], enhancing the loading of RNA polymerase II [
43] and is associated with increased soluble CD14 levels in healthy individuals [
44,
45]. Membrane-bound CD14 forms functional complexes with TLR2 or TLR4 and leukocyte β2-integrins, which could mediate TLR dimerization and optimize the innate immune response to MSU crystals [
10].
Acknowledgements
This work was supported by the Health Research Council of New Zealand, Arthritis New Zealand, New Zealand Lottery Health and the University of Otago. The authors would like to thank Jill Drake (Canterbury District Health Board), Roddi Laurence, Chris Franklin, Meaghan House (all University of Auckland) and Gabrielle Sexton (University of Otago) for recruitment. Matthew Brown, Linda Bradbury and The Arthritis Genomics Recruitment Initiative in Australia network are acknowledged. The European Crystal Network was formed after the first European Crystal Workshop in Paris, March 2010 (Prof Frédéric Lioté, Paris, and Prof Alexander So, Lausanne, convenors). The Atherosclerosis Risk in Communities Study is carried out as a collaborative study supported by National Heart, Lung, and Blood Institute contracts N01-HC-55015, N01-HC-55016, N01-HC-55018, N01-HC-55019, N01-HC-55020, N01-HC-55021, N01-HC-55022, R01HL087641, R01HL59367 and R01HL086694; National Human Genome Research Institute contract U01HG004402; and National Institutes of Health contract HHSN268200625226C. The authors thank the staff and participants of the ARIC study for their important contributions. Infrastructure was partly supported by Grant Number UL1RR025005, a component of the National Institutes of Health and NIH Roadmap for Medical Research. The Framingham Heart Study and the Framingham SHARe project are conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with Boston University. The Framingham SHARe data used for the analyses described in this manuscript were obtained through dbGaP. This manuscript was not prepared in collaboration with investigators of the Framingham Heart Study and does not necessarily reflect the opinions or views of the Framingham Heart Study, Boston University, or the NHLBI.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
CM, RKT and TRM helped to design the study, oversee its execution, and prepare the manuscript. LKS, ND, ROD, DRWK, KMW, MJ, TLJ, LAJ, TRR, PLR, A-KT, FL and AS helped to provide clinical recruitment and prepare the manuscript. All authors read and approved the final manuscript.