Article | Published:

Sarcoidosis is associated with a truncating splice site mutation in BTNL2

  • A Corrigendum to this article was published on 01 June 2005


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Change history

  • 17 May 2005

    Supplementary Table 3 online has been replaced


  1. 1.

    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.


  1. 1

    Newman, L.S., Rose, C.S. & Maier, L.A. Sarcoidosis. N. Engl. J. Med. 336, 1224–1234 (1997).

  2. 2

    Ziegenhagen, M. & Müller-Quernheim, J. The cytokine network in sarcoidosis and its clinical relevance. J. Intern. Med. 253, 18–30 (2003).

  3. 3

    Rybicki, B.A. et al. Familial aggregation of sarcoidosis. A case-control etiologic study of sarcoidosis (ACCESS). Am. J. Respir. Crit. Care Med. 164, 2085–2091 (2001).

  4. 4

    Schurmann, M. et al. Results from a genome-wide search for predisposing genes in sarcoidosis. Am. J. Respir. Crit. Care. Med. 164, 840–846 (2001).

  5. 5

    Sato, H. et al. HLA-DQB1*0201: a marker for good prognosis in British and Dutch patients with sarcoidosis. Am. J. Respir. Cell. Mol. Biol. 27, 406–412 (2002).

  6. 6

    Foley, P.J. et al. Human leukocyte antigen-DRB1 position 11 residues are a common protective marker for sarcoidosis. Am. J. Respir. Cell. Mol. Biol. 25, 272–277 (2001).

  7. 7

    Rybicki, B.A. et al. The major histocompatibility complex gene region and sarcoidosis susceptibility in african americans. Am. J. Respir. Crit. Care Med. 167, 444–449 (2003).

  8. 8

    Rossman, M.D. et al. HLA-DRB1*1101: a significant risk factor for sarcoidosis in blacks and whites. Am. J. Hum. Genet. 73, 720–735 (2003).

  9. 9

    Grutters, J.C. et al. Increased frequency of the uncommon tumor necrosis factor -857T allele in British and Dutch patients with sarcoidosis. Am. J. Respir. Crit. Care. Med. 165, 1119–1124 (2002).

  10. 10

    Abdallah, A. et al. Inhibitor kappa B-alpha (IkappaB-alpha) promoter polymorphisms in UK and Dutch sarcoidosis. Genes Immunity 4, 450–454 (2003).

  11. 11

    Stammers, M., Rowen, L., Rhodes, D., Trowsdale, J. & Beck, S. BTL-II: a polymorphic locus with homology to the butyrophilin gene family, located at the border of the major histocompatibility complex class II and class III regions in human and mouse. Immunogenetics 51, 373–382 (2000).

  12. 12

    Krawczak, M., Reiss, J. & Cooper, D.N. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum. Genet. 90, 41–54 (1992).

  13. 13

    Long, M. & Deutsch, M. Association of intron phases with conservation at splice site sequences and evolution of spliceosomal introns. Mol. Biol. Evol. 16, 1528–1534 (1999).

  14. 14

    Rhodes, D.A., Stammers, M., Malcherek, G., Beck, S. & Trowsdale, J. The cluster of BTN genes in the extended major histocompatibility complex. Genomics 71, 351–362 (2001).

  15. 15

    Sharpe, A.H. & Freeman, G.J. The B7-CD28 superfamily. Nat. Rev. Immunol. 2, 116–126 (2002).

  16. 16

    Albrecht, M., Domingues, F.S., Schreiber, S. & Lengauer, T. Identification of mammalian orthologs associates PYPAF5 with distinct functional roles. FEBS Lett. 538, 173–177 (2003).

  17. 17

    Ikemizu, S. et al. Structure and dimerization of a soluble form of B7-1. Immunity 12, 51–60 (2000).

  18. 18

    Schwartz, J.C., Zhang, X., Fedorov, A.A., Nathenson, S.G. & Almo, S.C. Structural basis for co-stimulation by the human CTLA-4/B7-2 complex. Nature 410, 604–608 (2001).

  19. 19

    Stamper, C.C. et al. Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses. Nature 410, 608–611 (2001).

  20. 20

    Zhang, X., Schwartz, J.C., Almo, S.C. & Nathenson, S.G. Crystal structure of the receptor-binding domain of human B7-2: insights into organization and signaling. Proc. Natl. Acad. Sci. USA 100, 2586–2591 (2003).

  21. 21

    Bajorath, J. Structural biology of T-cell costimulatory proteins: New insights, more surprises. J. Mol. Graph. Model. 19, 619–623 (2001).

  22. 22

    Reiling, N., Blumenthal, A., Flad, H.D., Ernst, M. & Ehlers, S. Mycobacteria-induced TNF-alpha and IL-10 formation by human macrophages is differentially regulated at the level of mitogen-activated protein kinase activity. J. Immunol. 167, 3339–3345 (2001).

  23. 23

    Hampe, J. et al. A non-electrophoretic method for high-throughput HLA-DRB1 group genotyping. Biotechniques 36, 148–151 (2004).

  24. 24

    Morton, N.E. & Collins, A. Tests and estimates of allelic association in complex inheritance. Proc. Natl. Acad. Sci. USA 95, 11389–11393 (1998).

  25. 25

    Stenzel, A. et al. Patterns of linkage disequilibrium in the MHC region on human chromosome 6p. Hum. Genet. 114, 377–385 (2004).

  26. 26

    Walunas, T.L., Bakker, C.Y. & Bluestone, J.A. CTLA-4 ligation blocks CD28-dependent T cell activation. J. Exp. Med. 183, 2541–2550 (1996).

  27. 27

    Borriello, F. et al. B7-1 and B7-2 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. Immunity 6, 303–313 (1997).

  28. 28

    Collins, A.V. et al. The interaction properties of costimulatory molecules revisited. Immunity 17, 201–210 (2002).

  29. 29

    Agadjanyan, M.G. et al. Costimulatory molecule immune enhancement in a plasmid vaccine model is regulated in part through the Ig constant-like domain of CD80/86. J. Immunol. 171, 4311–4319 (2003).

  30. 30

    Zissel, G. et al. Human alveolar epithelial cells type II are capable of regulating T-cell activity. J. Investig. Med. 48, 66–75 (2000).

  31. 31

    Hoebe, K. et al. Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways. Nat. Immunol. 4, 1223–1229 (2003).

  32. 32

    Ueda, H. et al. Association of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 423, 506–511 (2003).

  33. 33

    Costabel, U. & Hunninghake, G.W. ATS/ERS/WASOG statement on sarcoidosis. Sarcoidosis Statement Committee. American Thoracic Society. European Respiratory Society. World Association for Sarcoidosis and Other Granulomatous Disorders. Eur. Respir. J. 14, 735–737 (1999).

  34. 34

    Statement on sarcoidosis. Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee. Am. J. Respir. Crit. Care Med. 160, 736–755 (1999).

  35. 35

    Hampe, J. et al. Evidence for a NOD2-independent susceptibility locus for inflammatory bowel disease on chromosome 16p. Proc. Natl. Acad. Sci. USA 99, 321–326 (2002).

  36. 36

    Hampe, J. et al. An integrated system for high throughput TaqMan based SNP genotyping. Bioinformatics 17, 654–655 (2001).

  37. 37

    Hunninghake, G.W., Gadek, J.E., Kawanami, O., Ferrans, V.J. & Crystal, R.G. Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage. Am. J. Pathol. 97, 149–206 (1979).

  38. 38

    Clayton, D. & Jones, H. Transmission/disequilibrium tests for extended marker haplotypes. Am. J. Hum. Genet. 65, 1161–1169 (1999).

  39. 39

    Kruglyak, L., Daly, M.J., Reeve Daly, M.P. & Lander, E.S. Parametric and nonparametric linkage analysis: a unified multipoint approach. Am. J. Hum. Genet. 58, 1347–1363 (1996).

  40. 40

    Krawczak, M. et al. Allelic association of the cystic fibrosis locus and two DNA markers, XV2c and KM19, in 55 German families. Hum. Genet. 80, 78–80 (1988).

  41. 41

    Legouis, R. et al. Basolateral targeting by leucine-rich repeat domains in epithelial cells. EMBO Rep. 4, 1096–1102 (2003).

  42. 42

    Rosenstiel, P. et al. TNF-alpha and IFN-gamma regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells. Gastroenterology 124, 1001–1009 (2003).

Download references


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.

Authors' Contributions

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.

Author information

Correspondence to Jochen Hampe.

Ethics declarations

Competing interests

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.

Supplementary information

Supplementary Fig. 1

Computationally derived domain architecture of the BTNL2 gene product. (PDF 427 kb)

Supplementary Fig. 2

Plot of the Kyte-Doolittle hydropathy index for BTNL2. (PDF 58 kb)

Supplementary Fig. 3

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)

Supplementary Fig. 4

Heat map of pair-wise D′ on chromosome 6p21 in the German population. (PDF 198 kb)

Supplementary Table 1

Subphenotypic composition of the different samples used in the study. (PDF 34 kb)

Supplementary Table 2

DRB1 analysis. (PDF 46 kb)

Supplementary Table 3

DQB1 and DPB1 analysis. (PDF 80 kb)

Supplementary Methods (PDF 69 kb)

Supplementary Note (PDF 36 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading

Figure 1: Graphical representation of the stage I SNP screen on chromosome 6p21.
Figure 2: Graphical representation of the stage II SNP screen of the combined 'basic' and 'extension' samples.
Figure 3: Effect of rs2076530 on splicing and protein structure.
Figure 4: Expression pattern of BTNL2, as analyzed by nested RT-PCR in the Human Multiple Tissue cDNA Panels (Clontech, a) and in THP-1 cells (native and after a 4-h incubation with 10 ng ml−1 TNF-α) and BAL cells of individuals with sarcoidosis and normal controls (b).
Figure 5: Subcellular localization of the long and truncated forms of BTNL2 protein.