Systemic sclerosis (SSc) is a connective tissue disease that affects 7–489 individuals per million worldwide and is characterized by the excess production of extracellular matrix molecules and fibrosis (1). Patients with SSc display skin sclerosis, obliterative microvasculopathy such as Raynaud's phenomenon, and multiorgan involvement. Severe complications of SSc sometimes develop, including interstitial lung disease, pulmonary hypertension, and renal crisis. These severe symptoms and complications of SSc result in a poor prognosis and a shortened lifespan (2, 3). No effective method for preventing or curing SSc has been established (4).
It is well known that SSc has genetic components (5); for example, a US study revealed that the incidence of SSc was much higher among the families of patients with SSc compared with the general population (6). Recent technologic developments enabled the use of genome-wide association studies (GWAS) to identify novel susceptibility loci for autoimmune diseases (7). GWAS of European patients with SSc revealed that CD247 (8), HLA (8), TNIP1, PSORS1C1, and RHOB (9) are susceptibility loci for SSc. In addition, another GWAS identified associations between IRF8, GRB10, and SOX5 and limited cutaneous SSc (lcSSc) in a European population (10). Furthermore, studies adopting a candidate gene approach based on subjecting genes to functional inference analysis led to the identification of STAT4 (11), IRF5 (12), TBX21 (13), NLRP1 (14), TNFSF4 (15), CD226 (16), BLK (17), and TNFAIP3 (18) as novel susceptibility genes for SSc in Europeans. SSc association studies in Japanese populations confirmed that STAT4 (19), IRF5 (20), and BLK (21) are associated with SSc and identified UBE2L3 as a susceptibility gene for diffuse cutaneous SSc (dcSSc) (22). An association between HLA and SSc was also detected in Asians (23). These findings suggest a clear overlap in the genetic background of SSc between different populations.
It is well known that susceptibility genes are shared by various autoimmune diseases (24). In fact, HLA (25), STAT4 (26), and TNFAIP3 (27,28), which are susceptibility genes for SSc, have also been reported to be associated with rheumatoid arthritis (RA). In addition, PTPN22, which was shown to be strongly associated with RA in a European population (29), showed a suggestive association with SSc in Europeans (30). The sharing of these susceptibility genes between RA and SSc raises the possibility that newly identified susceptibility genes for RA could also be susceptibility genes for SSc. Recently, a large Japanese consortium, the Genetic and Allied research in Rheumatic diseases Networking consortium, identified 9 novel susceptibility genes and 6 candidate susceptibility genes for RA using a meta-analysis of GWAS and replication studies (31). Four other genes, namely, HLA, PADI4, CCR6, and TNFAIP3, were also confirmed to display associations with RA. Here, we performed a 2-stage association study of Japanese patients with SSc, in which we genotyped these genes as candidate susceptibility loci.
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- PATIENTS AND METHODS
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- Supporting Information
Because SSc can lead to severe complications, poor quality of life, and shortened survival, clarifying the characteristics of SSc is important. Clarification of the disease would aid the search for novel therapeutic targets and the development of new therapeutic strategies. Detecting susceptibility genes using GWAS or a candidate gene approach would also help to uncover the pathophysiology underlying SSc.
Previous studies have revealed that more than 15 markers and loci are associated with SSc. However, the markers detected so far cannot fully explain the genetics of SSc, indicating that many susceptibility genes are yet to be identified. Because a relatively large proportion of RA susceptibility genes are shared by other autoimmune diseases (24), a candidate gene approach using novel markers observed in GWAS of RA is a fascinating way of identifying new SSc markers. In fact, some of the novel susceptibility markers for RA identified in the meta-analysis were shown to be susceptibility markers for systemic lupus erythematosus (SLE) and Graves' disease (31).
In the current study, we successfully identified 3 susceptibility genes for SSc in Japanese. No studies have identified PLD4 as an SSc-associated locus. The current study is also the first to detect TNFAIP3 and IRF8 as susceptibility genes for SSc in a Japanese population. The best-fit models for each association are shown in Supplementary Table 4, available on the Arthritis & Rheumatism web site at http://onlinelibrary.wiley.com/doi/10.002/art.37777/abstract.
It is conceivable that these 3 associations might have been obtained due to the overlap of RA and SSc. Even after excluding the patients with both RA and SSc based on physicians' reports, the significant associations for the 3 loci were still observed (Table 3). Information regarding rheumatoid factor (RF) and anti–citrullinated protein antibody (ACPA) was available for 371 SSc patients without RA and 65 SSc patients without RA, respectively, of whom 21.6% and 10.8% were positive for RF and ACPA, respectively. These prevalences are compatible with those previously observed in SSc patients without RA (35, 36). Moreover, we showed that the effect sizes and risk direction of the markers tested in this study were dissociated between SSc and RA. In addition, further stringent analysis comprising SSc patients without any autoimmune disease also showed the associations of the 3 loci. These results indicate that the associations of the 3 loci are not attributable to overlapping of RA or other diseases.
Although the associations of the ARID5B and CD83 loci with SSc did not reach a stringently significant level in the combined study, the tendencies toward an association with SSc displayed by rs10821944 in the ARID5B locus and rs12529514 in the CD83 region in the first study were maintained in the replication study. This indicates that these loci are potential susceptibility regions for SSc. Further replication studies are needed to address the associations of these 2 loci with SSc in a Japanese population.
Because TNFAIP3 was reported to be strongly associated with SSc in a European population (18), the significant associations detected in the combined study indicate that TNFAIP3 displays general associations with SSc that go beyond ethnic boundaries. In addition, rs6932056, which displayed a strong association with SSc in a European population (18), is in strong LD with rs5029939 (r2 = 0.85) in the Japanese population. SNP rs6932056 also displays strong LD with rs2230926, a missense mutation of TNFAIP3 (r2 = 0.85), in Japanese. The rs2230926 missense mutation leads to an amino acid alteration in the OTU (ovarian tumor) domain of the A20 protein, which is considered to result in decreased NF-κB signaling. Because we did not observe strong associations between rs6932056 and SSc in the replication study, it will be necessary to reexamine the association between TNFAIP3 and SSc using independent sample sets of Japanese patients with SSc, in spite of the significant associations detected in this study.
PLD4 is a recently reported member of the phospholipase family without phospholipase D activity. PLD4 is expressed in the spleen and early postnatal microglia in the white matter of mice (37). The phenotypes of Pld4-deficient mice have not been reported. In addition, little is known about the expression or distribution of PLD4 in humans. Although the functions of PLD4 are also poorly understood, it is known to be involved in the phagocytosis of microglia (38). The expression of PLD4 around the marginal zone in the spleen might support the functional involvement of PLD4 in immunologic systems. It is interesting that rs2841280, which alters an amino acid of PLD-4, is associated with SSc. Minor allele G of rs2841280 is associated in a protective manner. The impact of an amino acid alteration brought by rs2841280 on the effect of PLD-4 protein is not known.
When we analyzed the impact of the amino acid alteration using in silico analysis (SIFT software; http://sift.jcvi.org/), it was shown to result in a small effect. However, the association raises the possibility that this polymorphism leads functional modulation of PLD-4, and it is feasible to analyze the functional change of PLD-4 protein with rs2841280, using animal models of SSc. When we performed an in silico analysis of the effect of rs2841277 and rs2841280 on PLD4 expression, we did not detect any clear associations between the 2 genotypes and PLD4 transcription (P > 0.05) (39). Therefore, in spite of the association of these 2 mutations, it has not been confirmed whether one of these 2 polymorphisms is the causative mutation.
Although the detection of a P value less than 5 × 10−8 in a GWAS is stringent evidence of an association between a marker and a particular disease, the detection of suggestive associations between the PLD4 region and SSc in European GWAS would indicate that associations exist between PLD4 and SSc in other populations. However, when we examined the associations between the PLD4 locus or nearby loci and SSc in GWAS involving a European population, we did not detect any strong associations (P < 10−4) (8, 9). According to the HapMap database, the European population displays a higher risk allele frequency for rs2841277 than the Japanese population. In addition, the HapMap database also indicates that the LD block spanning PLD4, which includes rs2841277, is similar in Europeans and Japanese. Nevertheless, a European population did not show a strong association between PLD4 and SSc, suggesting that PLD4 has a stronger effect on autoimmune diseases in Japanese than in Europeans. There is also a possibility that these 2 polymorphisms are only markers, and that a rare variant in LD with the 2 markers affects disease onset. A rare causative variant might explain a different association of PLD4 with SSc between populations.
IRF8 was shown to be associated with SLE in a European population (40). Interferon regulatory factor 8 (IRF-8) protein is a transcription factor involved in the interferon pathway. The interferon pathway has been shown to be involved with a broad range of autoimmune diseases, including SSc (41). Thus, it is interesting that IRF5 and IRF8, both of which belong to the IRF family, displayed associations with SSc. Although a European GWAS of SSc patients revealed suggestive associations between the IRF4 locus and SSc, the results were not successfully replicated (8), indicating that the different functional roles of each IRF family molecule might influence the development of SSc. IRF8 promotes B cell differentiation; however, the roles and importance of B cells in skin fibrosis in SSc patients have not been established (42–44). IRF8 and its mutant variants are also known to be involved in the development of dendritic cells (45). Thus, the association between IRF8 and SSc might indicate the involvement of B cells and dendritic cells in the development of SSc.
When the patients with SSc were classified as having either lcSSc or dcSSc and subanalyses were performed, ARID5B, IRF8, and CD83 displayed stronger associations with one of the 2 phenotypes. However, the associations of these 3 markers with the phenotypes were not strong enough to provide convincing evidence of a clear distinction between the genetic backgrounds of the 2 SSc phenotypes. When the associations of the SSc subtypes with the other 13 markers in the first set were analyzed, no strong association was detected (P > 0.05). Other subanalyses of the susceptibility loci in the combined set did not show significant results between disease phenotypes, due to lack of power. Because classification according to disease phenotypes resulted in limited numbers of subjects in each subset, we conducted this subanalysis only in the combined set. The association between TNFAIP3 and ACAs should be confirmed in a large-scale association study.
Although GWAS are an extremely powerful way to detect novel susceptibility genes for diseases, GWAS of patients with SSc have been performed only in European populations. Our study detected strong evidence for the sharing of susceptibility genes between RA and SSc in a Japanese population. In addition, the current study indicated that a candidate gene approach based on the results of GWAS of other diseases that display pathologic signaling pathways or mechanisms similar to those associated with the disease being examined is an effective approach to identifying novel susceptibility genes.
It will be interesting to perform GWAS of Japanese patients with SSc and analyze the similarities and differences in the detected associations not only between Japanese and Europeans but also between Japanese patients with SSc and Japanese patients with RA.
- Top of page
- PATIENTS AND METHODS
- AUTHOR CONTRIBUTIONS
- Supporting Information
All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Terao had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Terao, Ohmura, Kawaguchi, Nishimoto, Kawasaki, Takehara, Furukawa, Kochi, Ota, Ikari, Sato, Tohma, Yamada, Yamamoto, Kubo, Yamanaka, Kuwana, Tsuchiya, Matsuda, Mimori.
Acquisition of data. Terao, Ohmura, Kawaguchi, Nishimoto, Kawasaki, Takehara, Furukawa, Kochi, Ota, Ikari, Sato, Tohma, Yamada, Yamamoto, Kubo, Yamanaka, Kuwana, Tsuchiya, Matsuda, Mimori.
Analysis and interpretation of data. Terao, Ohmura.