Abstract
Objective. We evaluated whether a single-nucleotide polymorphism (SNP) of the TRAF6 gene previously associated with systemic lupus erythematosus and rheumatoid arthritis may be a common risk factor for systemic sclerosis (SSc) and giant cell arteritis (GCA).
Methods. A total of 1185 patients with SSc, 479 patients with biopsy-proven GCA, and 1442 unrelated healthy controls of white Spanish origin were genotyped for the rs540386 variant using a specifically designed TaqMan© allele discrimination assay.
Results. No significant associations of this SNP with global SSc or GCA were found. This was also the case when the potential associations of the TRAF6 polymorphism with the main clinical phenotypes of the 2 diseases (e.g., limited cutaneous and diffuse cutaneous SSc, or presence of polymyalgia rheumatica and visual ischemic manifestations in GCA) were assessed.
Conclusion. Our data do not support a role of the rs540386 TRAF6 variant as a key component of the genetic network underlying SSc and GCA.
Autoimmune diseases are complex multifactorial disorders caused by a combination of environmental and genetic factors, each with generally modest effects independently, that lead to an imbalance of the immune system1. Accumulating knowledge suggests a shared genetic basis underlying autoimmunity, and the hypothesis that a common network of genetic risk variants may influence the development of different autoimmune diseases is gaining interest2.
Recent studies have reported an association between the tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) gene on 11p12 and rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)3,4. TRAF proteins are cytoplasmic adapter molecules with a pivotal role in the immune response that have been shown to interact with numerous members of the TNF receptor family, although TRAF6 is also involved in the regulation of other receptors, including interleukin 1R, interleukin 18R, and Toll-like receptors (TLR)5.
To evaluate the possible involvement of TRAF6 in the pathogenesis of general autoimmunity, we analyzed whether an intronic single-nucleotide polymorphism (SNP) of this gene that has been associated with SLE and RA is also involved in other autoimmune diseases; these included systemic sclerosis (SSc), a chronic fibrotic autoimmune disorder with a genetic background similar to that in SLE6, and giant cell arteritis (GCA), a vasculitis characterized by inflammatory lesions of medium- and large-size arteries that shares some genetic associations with RA7.
MATERIALS AND METHODS
Study population
A white Spanish cohort of 1185 patients with SSc, 483 patients with GCA, and 1442 unrelated healthy controls was analyzed. All patients fulfilled the respective American College of Rheumatology criteria for each disease8,9. Additionally, the GCA condition was confirmed by a positive temporal artery biopsy. The Spanish National DNA Bank provided the control samples, which had the same criteria for sex and geographic origin. Our study was approved by the local ethical committees and informed written consent was obtained from all participants. Clinical features of the patients have been described previously10,11.
SSc subgroups were established based on the extent of skin involvement and autoantibody status as limited cutaneous SSc (lcSSc), diffuse cutaneous SSc (dcSSc), positive for anticentromere antibodies (ACA), and positive for antitopoisomerase antibodies (ATA), as well as for the presence of pulmonary fibrosis12. GCA subsets were established according to the presence/absence of polymyalgia rheumatica, visual ischemic manifestations, severe ischemic manifestations (comprising visual manifestations, cerebrovascular accidents, jaw claudication, or limb claudication of recent onset), and irreversible occlusive disease (if patients experienced at least 1 of the following complications: permanent visual loss, stroke, or limb claudication of recent onset), as described10.
Genotyping methods
DNA was extracted from peripheral blood cells using standard procedures. All participants were genotyped for the TRAF6 variant rs540386 using a predesigned TaqMan© allele discrimination assay (ID: C 2408956_10) in a 7900HT Fast Real-time polymerase chain reaction system (Applied Biosystems, Foster City, CA, USA).
Statistical analyses
PLINK (v1.07) software (Harvard University, Cambridge, MA, USA; http://pngu.mgh.harvard.edu/purcell/plink/) was used to construct 2 × 2 contingency tables and chi-square test and/or Fisher’s exact test, when necessary. OR and 95% CI were obtained according to Woolf’s method. P values < 0.05 were considered statistically significant.
RESULTS
The overall statistical power of the study is shown in Table 1. No deviation from Hardy-Weinberg equilibrium was observed (p = 0.05).
We first investigated the possible implication of the rs540386 TRAF6 variant in the genetic susceptibility to SSc and GCA and their major clinical subphenotypes by comparing the allele frequencies of the different case sets with that of the control population (Table 2). No statistical significance was observed for the global disease analyses (SSc vs controls: p = 0.39, OR = 0.93; GCA vs controls: p = 0.94, OR = 0.99) or when the different clinical subgroups were tested.
To further examine the potential role of the rs540386 genetic variant in SSc and GCA, a new comparison between patients positive for each specific clinical characteristic and patients without the corresponding manifestation was performed (Table 3). These analyses yielded similar negative results.
Finally, no significant heterogeneity between cases and controls was detected when the genotype, recessive, and dominant models were applied (Table 4).
DISCUSSION
TRAF6 is a ubiquitin ligase that mediates signal transduction pathways from the TNF and interleukin 1/TLR superfamilies, which implies that this protein is an important regulator of a wide spectrum of physiological processes including innate and adaptive immunity. TRAF6 is a key component of B cell activation and it has been reported that development of regulatory T cells (Treg), which are crucial in the maintenance of immune tolerance, requires TRAF6 expression in thymocytes13,14. TRAF6 has been associated with SLE and RA; members of the TLR signaling pathway upstream and downstream of TRAF6, such as TNFAIP3, IRF5, IRF7, and IRAK1, are known risk factors for SSc and other autoimmune diseases1,3,4,6.
Taking this into account, we considered TRAF6 an interesting candidate gene that could be involved in the predisposition to general autoimmunity. However, our data show no significant association of the analyzed polymorphism with the susceptibility and main clinical manifestations of SSc and GCA, although this same SNP has been described as strongly associated with SLE and RA3,4. Since the cohorts in our study were large and well defined, it is unlikely that the observed lack of association might have been due to a type II error as a consequence of low statistical power. Supporting this assumption, data from a genome-wide association study (GWAS) of SSc from our group15 found no positive association signals within the TRAF6 genomic region, which included 15 SNP (most of them closely linked with rs540386 in the CEU population of the HapMap project, Figure 1). However, no GWAS data are available for GCA, and replication in other populations, desirably of white origin, would be needed to draw definitive conclusions. Moreover, the possibility exists that a different TRAF6 SNP from rs540386 could be associated with SSc and GCA, as reported recently in SLE by Namjou, et al4.
Cumulative evidence indicates that the different clinical autoimmune outcomes would be a consequence of the presence in the genome of a set of common and disease-specific susceptibility loci interacting with epigenetic and environmental triggers2. In this regard, an increasing number of genetic loci outside the HLA region have been convincingly associated with a diverse range of autoimmune diseases, such as PTPN22, TNFAIP3, STAT4, CTLA4, IRF5, IL23R, IL2/IL21, and IL2RA, among others1. Our results suggest that TRAF6, or at least the SLE- and RA-associated rs540386 genetic variant, may not be a shared autoimmunity locus but a susceptibility factor specifically associated with the pathogenesis of certain autoimmune diseases. Further studies are needed to elucidate the extent of the common genetic contribution to autoimmunity.
Acknowledgment
The authors thank Sofía Vargas, Sonia García, and Gema Robledo for excellent technical assistance, and all the patients and controls for their essential collaboration. Banco Nacional de ADN (University of Salamanca, Spain) is thanked for supplying the control material.
Appendix
List of study collaborators. Members of the Spanish Scleroderma Group: Norberto Ortego-Centeno and Raquel Ríos, Unidad de Enfermedades Sistémicas Autoinmunes, Department of Internal Medicine, Hospital Clínico Universitario San Cecilio, Granada; Nuria Navarrete, Department of Internal Medicine, Hospital Virgen de las Nieves, Granada; Rosa García Portales, Department of Rheumatology, Hospital Virgen de la Victoria, Málaga; María Teresa Camps, Department of Internal Medicine, Hospital Carlos Haya, Málaga; Antonio Fernández-Nebro, Department of Rheumatology, and María F. González-Escribano, Department of Immunology, Hospital Virgen del Rocío, Sevilla; Julio Sánchez-Román, Francisco José García-Hernández, and M. Jesús Castillo, Department of Internal Medicine, Hospital Virgen del Rocío, Sevilla; M. Ángeles Aguirre and Inmaculada Gómez-Gracia, Department of Rheumatology, Hospital Reina Sofía, Córdoba; Benjamín Fernández-Gutiérrez, Department of Rheumatology, Hospital Clínico San Carlos, Madrid; Esther Vicente, Department of Rheumatology, Hospital La Princesa, Madrid; José Luis Andreu and Mónica Fernández de Castro, Department of Rheumatology, Hospital Puerta del Hierro, Madrid; Paloma García de la Peña, Department of Rheumatology, Hospital Madrid Norte Sanchinarro, Madrid; Francisco Javier López-Longo and Lina Martínez, Department of Rheumatology, Hospital General Universitario Gregorio Marañón, Madrid; Vicente Fonollosa, Department of Internal Medicine, Hospital Valle de Hebrón, Barcelona; Gerard Espinosa, Department of Internal Medicine, Hospital Clinic, Barcelona; Iván Castellví, Department of Rheumatology, Hospital Sant Pau, Barcelona; Carlos Tolosa, Department of Internal Medicine, Hospital Parc Tauli, Sabadell; Anna Pros, Department of Rheumatology, Hospital Del Mar, Barcelona; Mónica Rodríguez Carballeira, Department of Internal Medicine, Hospital Universitari Mútua Terrasa, Barcelona; Francisco Javier Narváez, Department of Rheumatology, Hospital Universitari de Bellvitge, Barcelona; Bernardino Díaz, Luis Trapiella, and María Gallego, Department of Internal Medicine, Hospital Central de Asturias, Oviedo; María del Carmen Freire and Inés Vaqueiro, Unidad de Trombosis y Vasculitis, Department of Internal Medicine, Hospital Xeral-Complexo Hospitalario Universitario de Vigo, Vigo; María Victoria Egurbide, Department of Internal Medicine, Hospital de Cruces, Barakaldo; Luis Sáez-Comet, Department of Internal Medicine, Hospital Universitario Miguel Servet, Zaragoza; Federico Díaz and Vanesa Hernández, Department of Rheumatology, Hospital Universitario de Canarias, Tenerife; Emma Beltrán, Department of Rheumatology, Hospital del Doctor Peset Aleixandre, Valencia; José Andrés Román-Ivorra, Department of Rheumatology, Hospital Universitari i Politecnic La Fe, Valencia; Francisco J. Blanco García, María Ángeles Robles, and Natividad Oreiro, Department of Rheumatology, INIBIC-Hospital Universitario A Coruña, La Coruña, Spain.
Members of the Spanish GCA Group: José A. Miranda-Filloy, Department of Rheumatology, Hospital Xeral-Calde, Lugo; Inmaculada C. Morado, Department of Rheumatology, Hospital Clínico San Carlos, Madrid; Javier Narváez and Carmen Gómez-Vaquero, Department of Rheumatology, Hospital Universitario de Bellvitge-IDIBELL, L’Hospitalet de Llobregat, Barcelona; Bernardo Sopeña, Thrombosis and Vasculitis Unit – Internal Medicine Department, Complejo Hospitalario Universitario de Vigo, Vigo; Ainhoa Unzurrunzaga, Department of Internal Medicine, Hospital de Galdakano, Vizcaya; Begoña Marí-Alfonso, Department of Internal Medicine, Corporació Sanitaria Parc Taulí, Instituto Universitario Parc Taulí, UAB, Sabadell, Barcelona; Eugenio de Miguel, Department of Rheumatology, Hospital Universitario de La Paz, Madrid; Ana Hidalgo-Conde, Department of Internal Medicine, Hospital Universitario Virgen de la Victoria, Málaga; Julio Sánchez, Department of Rheumatology, Hospital 12 de Octubre, Madrid; María J. García, Department of Rheumatology, Hospital Ramón y Cajal, Madrid.
Footnotes
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F.D. Carmona and A. Serrano share first authorship of this report; J. Martín and M.A. González-Gay share senior authorship.
- Accepted for publication March 7, 2012.