doi: 10.1111/j.1744-313X.2006.00580.x © 2006 Blackwell Publishing Ltd, International Journal of Immunogenetics 33, 155–161 155 Blackwell Publishing Ltd A polymorphism in the promoter region of the CD86 (B7.2) gene is associated with systemic sclerosis A. M. Abdallah,* E. A. Renzoni,* S. Anevlavis,* A. L. Lagan,* F. M. Munkonge,† C. Fonseca,‡ C. M. Black,‡ D. Briggs,§ A. U. Wells,* S. E. Marshall,* N. McHugh,¶ R. M. du Bois* & K. I. Welsh* Summary Systemic sclerosis (SSc) is a connective tissue disease of unknown aetiology characterized by fibrosis of the skin and internal organs, vascular abnormalities and humoral auto- immunity. Strong T-cell-dependent autoantibody and HLA associations are found in SSc subsets. The co-stimulatory molecule, CD86, expressed by antigen-presenting cells, plays a crucial role in priming naïve lymphocytes. We hypothesized that SSc, or one of the disease subsets, could be associated with single-nucleotide polymorphisms of the CD86 gene. Using sequence specific primer- polymerase chain reaction (SSP-PCR) methodology, we assessed four CD86 polymorphisms in 221 patients with SSc and 227 healthy control subjects from the UK. Haplotypes were constructed by inference and confirmed using PHASE algorithm. We found a strong association between SSc and a specific haplotype (haplotype 5), which was more prevalent in patients than in controls (29% vs 15%, OR = 2.3, χ 2 = 12, P = 0.0005). This association could be attributed to the novel -3479 promoter poly- morphism; a significant difference was observed in the distribution of the CD86 -3479 G allele in patients with SSc compared to controls (43.7% vs. 32.4%, OR = 1.7, χ 2 = 12.1, P = 0.0005). TRANSFAC analyses suggest that the CD86-3479T allele contains putative GATA and TBP sites, whereas G allele does not. We assessed the relative DNA protein-binding activity of the -3479 polymor- phism in vitro using electromobility gel shift assays (EMSA), which showed that the -3479G allele has less binding affinity compared to the T allele for nuclear proteins. These findings highlight the importance of co- stimulatory pathways in SSc pathogenesis. Introduction Systemic sclerosis (SSc) is a connective tissue disease of unknown aetiology characterized by fibrosis of the skin and internal organs, vascular abnormalities and humoral autoimmunity (Legerton, III et al ., 1995). With no defini- tive evidence supporting any single environmental cause, recent attention has focused on genetic factors, particu- larly genes at the interface of host and environment inter- action, as even minor variation in these genes could be critical for host defence or inflammatory response (Laza- rus et al ., 2002). Genetic predisposition to SSc is sug- gested by familial studies (Englert et al ., 1999; Arnett et al ., 2001), case-control association studies (Sato et al ., 2002, 2004) and animal models (Sgonc, 1999). Several lines of evidence suggest that T-cell activation plays a crucial role in the pathogenesis of SSc. Early in the course of the disease, T cells infiltrate the skin and are characterized by oligoclonal expansion, suggesting a response to as yet unidentified antigen/s (Sakkas et al ., 2002). Increased numbers of T cells are also found in the lungs of SSc patients with lung fibrosis (Wells et al ., 1995). Peripheral blood T cells show signs of activation, express- ing IL-2R, HLA-DR and CD29 (Freundlich & Jimenez, 1987). T-cell help is needed for SSc autoantibody produc- tion, as observed for antitopoisomerase antibody produc- tion (Kuwana et al ., 1995). T lymphocytes require two signals for full activation. The first signal is antigen-specific and based on recogni- tion of a peptide/major histocompatibility complex (MHC) by the T-cell receptor (TCR). The second signal is antigen non-specific and derives from the interaction between co-stimulatory molecules, including B7 family members CD80 (B7.1) and CD86 (B7.2), expressed on the cell surface of antigen-presenting cells, and the CD28 mole- cule on T cells (Sharpe & Freeman, 2002). This engage- ment is crucial to increase and sustain the T-cell response initiated by antigen-receptor signalling. We hypothesized that polymorphisms in the CD86 gene could modulate gene expression or function or both and influence the intensity of immune responses, contributing to suscepti- bility to SSc or disease severity. Four single-nucleotide polymorphisms (SNP), including one novel potentially functional SNP in the promoter region, have been identi- fied in the CD86 gene. The aims of our study were to * Clinical Genomics Group, † Department Gene Therapy, National Heart and Lung Institute, Imperial College London, ‡ Royal Free Hospital and University College London, § Department of Histocompatibility and Immunogenetics, National Blood Service, Birmingham, ¶ Royal National Hospital for Rheumatic Diseases, Centre for Rheumatology, Bath, UK Received 9 November 2005; accepted 22 December 2005 Correspondence: K. I. Welsh, Clinical Genomics Group, National Heart and Lung Institute, Imperial College London, London, UK. Tel: 0207-351-8354; Fax: 0207-351-8336; E-mail: k.welsh@imperial.ac.uk