Association of the C8orf13-BLK region with systemic sclerosis in North-American and European populations
Introduction
Scleroderma (systemic sclerosis, SSc) is a chronic, multisystem autoimmune disease clinically characterized by progressive fibrosis of the skin and internal organs. Pathologically, SSc exhibits three cardinal features: inflammation and autoimmunity, vasculopathy and excessive extracellular matrix production and deposition. How the disease process is triggered, remains to be established, but current paradigms point towards immune dysregulation as a central process in the pathogenesis of SSc.
Multiple lines of evidence, in patients as well as animal models of dermal fibrosis, point to dysregulation of the immune system and autoimmunity in the pathogenesis of SSc. Alterations in T-cell profiles and cytokines have been demonstrated [1]. In addition, the B-cell lineage is implicated [1], [2]. CD19 deficient mice have reduced dermal fibrosis after subcutaneous bleomycin administration [3]. The presence of multiple SSc-associated autoantibodies, most commonly anti-centromere (ACA), anti-topoisomerase Q1 antibodies (ATA), and anti-RNA polymerase III antibodies (ARA), has been well-described in SSc patients and provides indirect evidence for the importance of the B-cell lineage in SSc [4], [5], [6]. Interestingly, the SSc-associated autoantibodies correlate with distinct clinical subsets characterized by extent of cutaneous involvement and pattern of organ involvement [7], [8]. For example, pulmonary arterial hypertension is more common in patients with ACA, pulmonary fibrosis is more common in patients with ATA, and scleroderma renal crisis is more common in patients with ARA [7].
Recently, several genetic polymorphisms have been associated with SSc in susceptibility [9], [10], [11], [12], [13], [14], [15]. Many of these genes are susceptibility factors for other autoimmune diseases suggesting that they are common autoimmune disease susceptibility genes. These data support the paradigm of common dysregulated pathways across multiple autoimmune diseases. Interestingly, some of these genetic variants associated with SSc are associated with susceptibility to developing SSc, while others increase risk only in specific autoantibody subsets seen in SSc [9], [10].
Two genome wide association studies in systemic lupus erythematosus (SLE) have implicated the C8orf13-BLK (B lymphoid tyrosine kinase gene) region of chromosome 8p23.1 as a susceptibility locus for SLE [16], [17]. These findings were further replicated in a Japanese population [18]. B lymphoid kinase (Blk), encoded by the BLK gene is a member of the Src family kinases (SFKs) which includes Blk, Lck, Fyn, Lyn, c-Src, c-Yes, Fgr, and Hck [19]. Blk is the only SFK that is exclusively expressed in B cells and thymocytes and not in mature T cells [20], [21], [22]. Blk transduces signals downstream of the B cell receptor (BCR) and plays a role in BCR signaling and B cell development [23], [24]. B cell development is dependent upon the activation of the transcription factor nuclear factor κB (NFκB) by SFKs (Blk, Fyn, Lyn) [23]. The C8orf13 gene is ubiquitously expressed but its exact function is currently unknown.
Given the importance of B-cells and autoantibodies in the pathogenesis of SSc as well as SLE and the emerging paradigm that autoimmune diseases may share common susceptibility genes, the current study sought to investigate the potential association of the C8orf13-BLK region variants with SSc in two large, independent, and well-described case–control series. Herein we demonstrate a significant association of both C8orf13-BLK region variants with susceptibility to SSc and more strongly with the anti-centromere antibody subset of SSc in both case–control series. We observe a dysregulation of the B-cell receptor and NFκB signaling based on the risk haplotype of these variants in peripheral blood gene expression arrays.
Section snippets
SSc patients and controls
In this study, we combined 1050 patients of North Americans of European descent with SSc and 694 unrelated healthy controls of North Americans of European descent from the Scleroderma Family Registry and DNA Repository, the Genetics versus Environment in Scleroderma Outcomes Study (GENISOS), and from SSc patients evaluated in the University of Texas Rheumatology Division, dating from 1986 to present, as previously described [8], [25]. For a replication cohort, an independent Spanish
SNP genotyping, linkage disequilibrium, and haplotype block structure
The C8orf13-BLK gene region variants (rs13277113–rs2736340) were in Hardy–Weinberg Equilibrium in both the North American and Spanish control samples. CEPH DNA samples were used as controls to improve the accuracy of allele calling on Taqman 5′ genotyping assay. The SNP call rate for both the SNPs in both the case–control series was greater than 97%. Pairwise LD was calculated by both D′ and r2 between rs13277113 and rs2736340 variants in both the control series. The SNPs demonstrated strong LD
Discussion
Herein, we report the association of SSc with two variants (rs13277113 and rs2736340) in the C8orf13-BLK gene region in case–control subjects from North Americans of European descent and confirm this association in a Spanish case–control series. Studying a combined 1639 SSc patients and 1416 healthy controls we were able to demonstrate an additive effect of these variants for susceptibility to SSc. Furthermore, pathway analysis using the whole blood mRNA gene expression arrays highlighted
Acknowledgments
This study was supported by the NIH/NIAMS Center of Research Translation (CORT) in Scleroderma (P50AR054144) (Dr. Arnett), the NIH/NIAMS Scleroderma Family Registry and DNA Repository (N01-AR-0-2251) (Dr Mayes), UTHSC-H Center for Clinical and Translational Sciences (Houston CTSA Program) (NIH/NCRR 3UL1RR024148) (Dr. Arnett). This study was also supported by grants SAF2006-00398, CTS-1180 and GENFER (Dr. Martin). B.R. is supported by the I3P CSIC program funded by the “Fondo Social Europeo”.
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These authors contributed equally to this manuscript.