Common variants at TRAF3IP2 are associated with susceptibility to psoriatic arthritis and psoriasis.

Psoriatic arthritis (PsA) is an inflammatory joint disease that is distinct from other chronic arthritides and which is frequently accompanied by psoriasis vulgaris (PsV) and seronegativity for rheumatoid factor. We conducted a genome-wide association study in 609 German individuals with PsA (cases) and 990 controls with replication in 6 European cohorts including a total of 5,488 individuals. We replicated PsA associations at HLA-C and IL12B and identified a new association at TRAF3IP2 (rs13190932, P = 8.56 × 10⁻¹⁷). TRAF3IP2 was also associated with PsV in a German cohort including 2,040 individuals (rs13190932, P = 1.95 × 10⁻³). Sequencing of the exons of TRAF3IP2 identified a coding variant (p.Asp10Asn, rs33980500) as the most significantly associated SNP (P = 1.13 × 10⁻²⁰, odds ratio = 1.95). Functional assays showed reduced binding of this TRAF3IP2 variant to TRAF6, suggesting altered modulation of immunoregulatory signals through altered TRAF interactions as a new and shared pathway for PsA and PsV.

Psoriatic arthritis (PsA) is an inflammatory joint disease distinct from other chronic arthritides and frequently accompanied by psoriasis vulgaris (PsV) and seronegativity for rheumatoid factor. In a SNP array based genome wide association study (GWAS) of a German case/control collective, we confirmed HLA-C and IL12B as PsA susceptibility genes and found and replicated association to intragenic variants of TRAF3IP2 in various European study groups (1.39×10−12 > p > 8.56×10−17). These SNPs were also associated in a German psoriasis vulgaris cohort. Sequencing revealed coding variant p.D10N (rs33980500) as strongly associated (p = 1.13×10−20, OR = 1.95) and the only variant present on all risk haplotypes. Association with PsV was weaker (p = 1.35×10−4), although we are currently unable to establish its primary association with PsA. Functional assays for interaction showed reduced binding of this but not other TRAF3IP2 variants to TRAF6 suggesting altered modulation of immunoregulatory signals via altered TRAF-interactions as a novel pathway for PsA and PsV.

Psoriasis vulgaris (PsV) is a common chronic inflammatory disease that affects the skin and is mediated by T-cells. The more than coincidental occurrence of PsV with an inflammatory arthritis in up to 15 % of PsV patients 1 led to the recognition of psoriatic arthritis (PsA) as a separate disease entity. In order to distinguish PsA from other forms of inflammatory arthritis, consensus classification criteria (CASPAR) were recently established 2. A strong genetic component has been suggested by several studies, indicated for example by a relative risk of up to 40 for first degree and 12 for second degree relatives 3, which suggests a stronger heritability than PsV 4. However, knowledge of the genetic components contributing to PsV is more advanced than in PsA. Common established susceptibility factors for PsV and PsA are an HLA-C risk allele 5,6 as well as variants in/ near the IL23R and IL12B genes 7,8, while a frequent deletion within the epidermal differentiation complex contributes to skin (PsV) 9, but not to joint involvement (PsA) 10.

In order to identify additional susceptibility genes for PsA, we performed a genome wide association study (GWAS) in 609 German PsA patients and 990 control individuals using Affymetrix Genome-Wide Human SNP Array 6.0. After genotyping, stringent quality control and imputation of SNPs in linkage disequilibrium (LD), 1,585,307 SNPs in 572 cases and 888 controls were used in the analysis. P-values corrected for inflation showed a final λGC = 1.055 (see online methods). This post-quality-control (QC) sample size had 80% power to detect an OR of 1.63 for an allele frequency of 0.5 or an OR of 2.20 for an allele frequency of 0.1 at genome-wide significance level, taking into account the final λGC. A quantile-quantile plot of the initial GWAS data showed substantial deviation from the expected distribution of genome-wide p-values for an unusually high number of SNPs (Supplementary Fig. 1a) which was considerably reduced upon removal of SNPs within the MHC region (Supplementary Fig. 1b).

We initially identified genome-wide significant association (p < 5×10−8) at two well established susceptibility loci (Fig. 1, Table 1): HLA-C indicated by the upstream variant rs13191343 (p = 2.63×10−23) and IL12B indicated by the upstream variant rs12188300 (p = 5.60×10−13). At a third locus, two intronic variants (rs13196377, rs13210247) as well as one coding variant (rs13190932) of the TRAF3IP2 gene, not previously reported to be involved in PsA susceptibility, were significantly associated (5.76×10−7 < p < 9.36×10−7) . In the replication phase, genome-wide significant association (p = 4.53×10−10) was also observed at this locus.

Figure 1

Genome wide association results from the initial GWAS analysis. Negative decadic logarithms of genomic control corrected EIGENSTRAT p-values are plotted against genomic position (hg18). (a) Plot of 1,585,307 SNPs (after imputation) in 572 German psoriatic arthritis patients versus 888 German controls. The red line represents the genome wide significance threshold of 5×10−8. (b) Regional association of the TRAF3IP2 region. Genotyped SNPs appear in red, imputed SNPs in blue. Recombination rate (from HapMap data) is indicated by the light blue graph.

Table 1

Association analyses of genome-wide significant SNPs. Number of alleles (risk/non-risk allele) and allele frequencies (risk allele) in GWAS, replication and combined study and results of Cochran-Mantel-Haenszel test as well as EIGENSTRAT corrected GWAS data are shown

SNP	Locus	AssociatedAllele	Study	No. ofAllelesCases	No. ofAllelesControls	AlleleFrequencyCases	AlleleFrequencyControls	p-Value(CMH)	p-Value(EIGENSTRAT)	Odds Ratio	95%ConfidenceInterval
rs13191343	HLA	T	GWAS	351/749	270/1442	0.319	0.158	2.63×10−23	2.25×10−20	2.48	2.06-2.97
			Replication	863/2545	866/6398	0.253	0.119	7.61×10−51		2.33	2.09-2.61
			Combined	1214/3294	1136/7840	0.269	0.127	2.32×10−72		2.37	2.16-2.61
rs12188300	IL12B	T	GWAS	157/917	108/1598	0.146	0.063	5.60×10−13	2.40×10−8	2.54	1.96-3.29
			Replication	374/3068	534/6248	0.109	0.079	5.14×10−8		1.50	1.29-1.73
			Combined	531/3985	642/7846	0.118	0.076	7.33×10−17		1.70	1.50-1.93
rs13196377	TRAF3IP2	A	GWAS	138/990	105/1649	0.122	0.060	5.48×10−9	9.36×10−7	2.17	1.66-2.82
			Replication	283/3167	421/6789	0.082	0.058	2.83×10−6		1.50	1.27-1.78
			Combined	421/4157	526/8438	0.092	0.059	1.39×10−12		1.67	1.45-1.93
rs13190932(p.R74W)	TRAF3IP2	A	GWAS	129/983	97/1641	0.116	0.056	1.03×10−8	9.19×10−7	2.19	1.66-2.88
			Replication	294/3180	381/6841	0.085	0.053	4.53×10−10		1.71	1.44-2.02
			Combined	423/4163	478/8482	0.092	0.053	8.56×10−17		1.83	1.59-2.12
rs13210247	TRAF3IP2	G	GWAS	142/1000	112/1664	0.124	0.063	1.49×10−8	5.76×10−7	2.08	1.61-2.69
			Replication	316/3152	440/6802	0.091	0.061	3.79×10−8		1.56	1.33-1.84
			Combined	458/4152	552/8466	0.099	0.061	1.73×10−14		1.69	1.48-1.94

In order to confirm association to these loci, validation groups comprising a total of 1,761 patients and 3,727 control subjects of European descent from the Psoriatic Arthritis Genetics in Europe (PAGE) Consortium were genotyped for the best SNPs at HLA-C and IL12B as well as for the three most significantly associated variants at TRAF3IP2. As expected, the most significant association was observed with the HLA region: rs13191343, located 1.2 kB upstream of the gene HLA-C, had the strongest evidence for association both in the initial GWAS and in the validation study (Table 1, Fig. 1b). Taking into account the differing European background of our study groups, we used the Cochran-Mantel-Haenszel test in order to determine the level of association in the GWAS data set, the validation groups and the combined data set. rs12191877 has previously been described as the most significant associated PsV variant in a GWAS of individuals of European origin 11 and is located further upstream (13kB) of HLA-C. Analysis of this SNP (data not shown) revealed strong LD with rs13191343 (r2=0.95) in German PsA cases, suggesting that these two signals are not independent. Similarly, we observed strong LD (r2 = 0.95) to the most significantly associated variant rs10484554 described in a combined study for PsA and PsV 12 that is even further upstream.

Association to rs12188300 at IL12B (Table 1, Supplementary Fig. 2c) was confirmed in the validation groups. This locus is a known PsV susceptibility locus and previous studies have identified two SNPs that contribute to risk to PsV, independently of each other (rs3212227 and rs6887695) 7. Association of both variants with PsA has also been reported previously 8,13. However, a so far unreported third SNP, rs12188300, located 72kB upstream of the gene, showed the most significant association in our PsA GWAS (Table 1 and Supplementary Table 1). This variant may represent an independent risk factor as its LD with rs2082412 was negligible (r2 = 0.02). It is located in a moderately conserved sequence that might modify expression of IL12B, but this will require experimental confirmation.

Association with the TRAF3IP2 gene was also confirmed in the independent validation groups and in a combined analysis the evidence for association was considerable (Table 1). This gene has not previously been implicated in any immunological disease. Two intronic variants (rs13210247, rs13196377) as well as one coding variant (rs13190932, p.R74W [NM_147686]) were convincingly associated with odds ratios (ORs) of up to 1.8 (Table 1). Comparative analyses of the three SNPs in German individuals with either PsA or an independent group with PsV suggested a weaker association with PsV than with PsA, even more so when compared to a subset of 788 PsV patients with disease duration of more than 15 years who are unlikely to develop PsA (Supplementary Table 2). Although ORs were higher for PsA, the 95% confidence intervals overlapped with those observed in PsV, so it remains possible that the different effect sizes between psoriasis phenotypes are due to chance and not because of a primary association with PsA. Interestingly, a GWAS of psoriasis samples published in this issue shows lower OR for a highly correlated SNP mapping to the locus 14, but further detailed analyses are necessary to determine whether it is indeed primarily associated with PsA. All three variants tag a haplotype present in 2-10 % of Europeans as well as individuals of African and Asian (Han Chinese / Japanese) descent. Further sequencing of all exons of TRAF3IP2 in 24 PsA patients revealed a further coding variant (rs33980500, p.D10N [NM_147686]). Genotyping in single individuals showed an even stronger association with PsA (pcombined = 1.13×10−20 , OR = 1.95) as well as association to PsV (p=1.35−4, OR=1.52). SNP rs33980500 (p.D10N) but not rs13190932 (p.R74W) is present also on an additional rarer haplotype (3.8%) associated with PsA and PsV (Fig. 2a and Suppl. Table 3), indicating that this is the true causative variant. Comparison with sequences of Chimpanzee, other mammals as well as Neanderthal indicates that the non-risk allele (D) is the ancestral one. Both coding variants lead to an amino acid substitution in the N-terminal TRAF binding site.

Figure 2

Haplotype and functional analysis of associated variants in TRAF3IP2. (a) Exon/intron structure of TRAF3IP2 with location of associated SNPs. The four most common haplotypes in a combined set of 2,077 PsA cases and 2,648 control individuals of European origin and their frequency within cases are given. Risk alleles are in bold type. For each haplotype, odds ratios and 95% confidence intervals (in brackets) compare occurrence of this vs. all three other haplotypes. (b) Interaction of wild type TRAF3IP2 (Act1) and PsA-associated variants D10N and R74W with TRAF6 in a mammalian two hybrid dual-luciferase reporter assay. Depicted are means of relative normalized luminescence units reflecting the specific induction of firefly-luciferase by the interaction of the cotransfected ligand pairs TRAF3IP2 and TRAF6 [blue bars]. The investigated TRAF3IP2-variants include constructs harboring either single missense variant (D10N or R74W) or both (D10N+R74W). To exclude autoactivation activity, each prey and bait construct was separately cotransfected with reporter plasmids into HEK293 cells in the absence of any potential interaction partner [open bars]. P53 andTRAF2 as a pair of non-interacting proteins served as an additional independent negative control. Error bars represent 95%-confidence intervals.

TRAF3IP2 codes for Act1, a signalling adaptor involved in the regulation of adaptive immunity. Studies of TRAF3IP2-deficient mice suggest that Act1 is a negative regulator of humoral immunity via its inhibitory effect on CD40- and BAFFR-mediated signalling 15-15. This B-cell-specific negative regulatory impact on the CD40 pathway by TRAF3IP2 15 as a newly proposed PsA-associated gene is consistent with the lack of an autoantibody signature in PsA contrary to rheumatoid arthritis (RA). On the other hand, Act1 concomitantly operates as a positive signalling adaptor in IL-17-mediated cellular immune responses 15,16. IL-17 is a dominant ‘signature’ cytokine of TH-17 cells and upregulates neutrophil-mobilizing cytokines, chemokines, and tissue-degrading matrix-metalloproteases 17. IL-17-dependent receptor ligation induces Act1-recruitment to the cytoplasmic tail of the IL-17R 15. This in turn allows the incorporation of TNF receptor-associated factors (TRAF) TRAF3 and TRAF6 into the signaling complex and the subsequent ‘downstream’ activation of the MAPK- and NF-κB pathway 15,16,18. Accordingly, Act1 is not only involved in pathways balancing humoral and cellular immunity, but also represents a chief link between IL-17-mediated adaptive immune responses and NF-κB as the master regulator of innate immunity 19 controlling the inducible transcription of various pro-inflammatory cytokines. Interestingly, recent GWAS for PsV and RA have identified different genes involved in NF-κB regulation as influencing susceptibility: TNFAIP3 for both diseases 11,20, TNIP1 for PsV 11 and PRKCQ and TNFRSF14 for RA 21,22. None of these, though, was significantly associated in our PsA study.

Given the position of the two coding TRAF3IP2-associated SNPs, we hypothesized that the associated alleles may affect interaction with TRAF3 and/or TRAF6. We therefore investigated binding of the alleles using a mammalian-two-hydrid dual-luciferase reporter assay 23 (Fig. 2b). In our investigations we detected a strong and specific firefly luciferase reporter induction upon the interaction between the cotransfected TRAF6-bait and wildtype TRAF3IP2-prey constructs (up to 15-40 fold of respective negative controls, Fig. 2b). Since the bait samples containing TRAF3 instead of TRAF6 remained negative with all cotransfected TRAF3IP2 prey variants (results not shown) in agreement with earlier mammalian two hydrid studies 23, we focused in our further functional analysis on the interaction between TRAF6 and wildtype as well as mutant TRAF3IP2. The introduction of the N-coding risk allele of rs33980500 (p.D10N) in the amino terminal domain of Act1 resulted in a nearly complete loss of ability to interact with TRAF6 as indicated by a luciferase reporter activity in the range of negative control. (Fig. 2b). By contrast, the PsA-associated TRAF3IP2-allele coding for W at rs13190932 (p.R74W) did not cause any detectable change of TRAF6-binding as indicated by luminescence signals not significantly different from those determined for TRAF6-interaction with wild type TRAF3IP2. When both PsA-risk alleles (p.D10N+p.R74W) were present in the same TRAF3IP2-construct, the negative effect of the p.D10N-mutation on TRAF6 interaction was dominant (Fig. 2b). One-way analysis of variance (ANOVA) showed that differences of the mean normalized luciferase responses are highly significant (p = 3.17×10−11 for the interaction with TRAF6). The p.D10N mutation of Act1 identified as critical for TRAF6 interaction is also present on both risk-conferring haplotypes of TRAF3IP2 (Fig. 2a, Supplementary Table 3). Accordingly, our results suggest that p.D10N may modulate downstream signals of different crucial immunoreceptors via altered TRAF-interactions. This may ultimately lead to a shift in balance between adaptive B- and T-cell-responses and innate immunity into an arthritogenic disequilibrium. The functional data, however, should be viewed as preliminary and in need of further confirmation. The delineation of the involved pathways and how that predisposes to PsA will require further functional studies.

In summary, our study provides new insights into the pathophysiology of PsA, highlighting a novel susceptibility gene and identifying a TRAF3IP2 coding variant which may affect binding of interacting proteins. Likely, many more variants of weaker effect are involved in PsA susceptibility but our study is underpowered to detect them. Further studies in larger cohorts and meta-analyses will be required to unravel these as well as studies on endophenotypes which might have stronger genetic determinants.

URLs

PLINK (vs. 1.07) available at: http://pngu.mgh.harvard.edu/purcell/plink/