Sarcoidosis is a polygenic immune disorder with predominant manifestation in the lung. Genome-wide linkage analysis previously indicated that the extended major histocompatibility locus on chromosome 6p was linked to susceptibility to sarcoidosis. Here, we carried out a systematic three-stage SNP scan of 16.4 Mb on chromosome 6p21 in as many as 947 independent cases of familial and sporadic sarcoidosis and found that a 15-kb segment of the gene butyrophilin-like 2 (BTNL2) was associated with the disease. The primary disease-associated variant (rs2076530; PTDT = 3 × 10−6, Pcase-control = 1.1 × 10−8; replication PTDT = 0.0018, Pcase-control = 1.8 × 10−6) represents a risk factor that is independent of variation in HLA-DRB1. BTNL2 is a member of the immunoglobulin superfamily and has been implicated as a costimulatory molecule involved in T-cell activation on the basis of its homology to B7-1. The G → A transition constituting rs2076530 leads to the use of a cryptic splice site located 4 bp upstream of the affected wild-type donor site. Transcripts of the risk-associated allele have a premature stop in the spliced mRNA. The resulting protein lacks the C-terminal IgC domain and transmembrane helix, thereby disrupting the membrane localization of the protein, as shown in experiments using green fluorescent protein and V5 fusion proteins.
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NOTE: In the version of Supplementary Table 3 initially published online, the nomenclature of DQB and DPB alleles was partly incorrect. The errors have now been corrected and Supplementary Table 3 online has been replaced. Neither the stratified analyses that highlighted the independence of the BTNL2 effect from the two HLA loci (Supplementary Table 3 online) nor any other conclusions of the manuscript were affected by these mistakes.
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We thank all affected individuals, families, physicians, the German Sarcoidosis Patient Organization (Deutsche Sarcoidose-Vereinigung e. V.) and the contributing pulmonary specialist physicians for their cooperation; M. Albrecht, T. Wesse, P. Petersen, T. Henke, S. Kröger and I. Gorlich for technical help; P. Croucher and S. Jenisch for discussions and providing reagents; C. Manaster for database support; and H. Brade for providing the LPS. This study was supported by the German National Genome Research Network, the German Human Genome Project, the German Research Council, the BioSapiens NoE of the EU, the POPGEN population project, Mucosaimmunologie Forschungsgesellschaft, Applied Biosystems and a SUR grant from IBM.
R.V. helped to develop the SNP, HLA-DRB1, HLA-DQB1 and HLA-DPB1 assays and carried out genotyping, mutation detection and basic data analysis. J.H. prepared the manuscript and performed the data analysis, experimental planning, strategy and method development. K.H. and M.P. carried out cDNA cloning and splice assays. P.R. and D.S. carried out expression analysis and transfection experiments. A.S. and A.K. helped to develop the SNP assay. M.N.: helped to design the HLA-DRB1, HLA-DQB1 and HLA-DPB1 assays. A.F. carried out genotyping of HLA-DRB1, HLA-DQB1 and HLA-DPB1. K.I.G. carried out BAL cDNA work. R.H. carried out expression analysis. M.K. helped with data analysis and preparation of the manuscript. M.A. and T.L. helped with preparation of the manuscript and in silico analysis. E.S., M.S. and J.M.Q. helped with experimental design, patient recruitment, clinical characterization and preparation of the manuscript. N.R. and S.E. carried out induction experiments in monocytes. S.S. helped with experimental strategy and preparation of the manuscript.
The University of Kiel (Germany) has filed a patent application based on the findings reported in this paper. Clearance of any published finding with the university patent office is mandatory.
Computationally derived domain architecture of the BTNL2 gene product. (PDF 427 kb)
Plot of the Kyte-Doolittle hydropathy index for BTNL2. (PDF 58 kb)
Real-time PCR analysis of BTNL2 expression in unstimulated THP cells and after incubation for 4 hours with 50ng/ml IL 1β and 10ng/ml TNF-α. (PDF 55 kb)
Heat map of pair-wise D′ on chromosome 6p21 in the German population. (PDF 198 kb)
Subphenotypic composition of the different samples used in the study. (PDF 34 kb)
DRB1 analysis. (PDF 46 kb)
DQB1 and DPB1 analysis. (PDF 80 kb)
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