Genes and Immunity (2010) 11, 279–293 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 $32.00 www.nature.com/gene ORIGINAL ARTICLE RGMA and IL21R show association with experimental inflammation and multiple sclerosis R Nohra1, AD Beyeen1, JP Guo2, M Khademi1, E Sundqvist1, MT Hedreul1, F Sellebjerg3, C Smestad4, AB Oturai3, HF Harbo4,5, E Wallstro¨m1, J Hillert6, L Alfredsson7, I Kockum1, M Jagodic1, J Lorentzen2 and T Olsson1 1Department of Clinical Neuroscience, Neuroimmunology Unit, Karolinska Institutet, Stockholm, Sweden; 2Department of Biochemistry and Biophysics, Medical Inflammation Research, Karolinska Institutet, Stockholm, Sweden; 3Danish Multiple Sclerosis Center Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark; 4Department of Neurology, Oslo University Hospital, Ulleva˚l, Oslo, Norway; 5Department of Neurology, Faculty Division Ulleva˚l, Oslo University Hospital, University of Oslo, Oslo, Norway; 6Department of Clinical Neuroscience, Division of Neurology, Karolinska Institutet, Stockholm, Sweden and 7Department of Cardiovascular Epidemiology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Rat chromosome 1 harbors overlapping quantitative trait loci (QTL) for cytokine production and experimental models of inflammatory diseases. We fine-dissected this region that regulated cytokine production, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), anti-MOG antibodies and pristane-induced arthritis (PIA) in advanced intercross lines (AILs). Analysis in the tenth and twelfth generation of AILs resolved the region in two narrow QTL, Eae30 and Eae31. Eae30 showed linkage to MOG-EAE, anti-MOG antibodies and levels of interleukin-6 (IL-6). Eae31 showed linkage to EAE, PIA, anti-MOG antibodies and levels of tumor necrosis factor (TNF) and IL-6. Confidence intervals defined a limited set of potential candidate genes, with the most interesting being RGMA, IL21R and IL4R. We tested the association with multiple sclerosis (MS) in a Nordic case–control material. A single nucleotide polymorphism in RGMA associated with MS in males (odds ratio (OR) ¼ 1.33). Polymorphisms of RGMA also correlated with changes in the expression of interferon-g (IFN-g) and TNF in cerebrospinal fluid of MS patients. In IL21R, there was one positively associated (OR ¼ 1.14) and two protective (OR ¼ 0.87 and 0.68) haplotypes. One of the protective haplotypes correlated to lower IFN-g expression in peripheral blood mononuclear cells of MS patients. We conclude that RGMA and IL21R and their pathways are crucial in MS pathogenesis and warrant further studies as potential biomarkers and therapeutic targets. Genes and Immunity (2010) 11, 279–293; doi:10.1038/gene.2009.111; published online 14 January 2010 Keywords: multiple sclerosis; experimental autoimmune encephalomyelitis; autoimmunity; RGMA; IL21R Introduction demonstrated for type 1 diabetes and MS genes.12 Therefore, cross-disciplinary genetics may be rewarding. Common inflammatory autoimmune disorders, such as Discovery of additional genes contributing to MS and multiple sclerosis (MS), type 1 diabetes and rheumatoid their disease regulatory mechanisms may allow the arthritis (RA) are complex chronic diseases with poorly development of more selective therapies and biomarkers understood etiologies. We are particularly interested in in MS. MS that is a chronic inflammatory disease of the central There are many obstacles in studying genetic regula- nervous system. Both environmental and genetic factors tion of autoimmune disorders in human cohorts, includ- contribute to its etiology.1 The human leukocyte antigen ing limited possibility of functional studies and an complex is a major genetic regulator of MS,2–4 whereas uncontrolled contribution of environmental factors. non-human leukocyte antigen genes are numerous and Positioning of disease regulating loci can also be have low odds ratios (ORs).5,6 Only recently, with achieved using animal models in rodents mimicking analysis of very large cohorts, non-human leukocyte the human diseases in which both genetic and environ- antigen MS genes are starting to be unambiguously mental factors can be controlled. Numerous quantitative identified.7–14 Another important concept is the sharing trait loci (QTL) have previously been mapped using of risk genes between inflammatory diseases,15 as now crosses between inbred rodent strains with diverse susceptibilities to autoimmune inflammatory diseases.16 Recent progress suggests that this strategy is productive Correspondence: Dr R Nohra, Department of Clinical Neuroscience, in revealing susceptibility genes and functional path- Neuroimmunology Unit, Neuroimmunology Unit, CMM, L8:04, ways shared between experimental models and complex Karolinska University Hospital, Stockholm SE-171 76, Sweden. 17 E-mail: [email protected] human disorders. Received 9 June 2009; revised 27 November 2009; accepted 30 Experimental autoimmune encephalomyelitis (EAE), November 2009; published online 14 January 2010 a model for MS, has defined pathogenic mechanisms RGMA and IL21R R Nohra et al 280 underlying neuroinflammation, and has allowed MOG-induced EAE (MOG-EAE)29 and in 465 (DA  18 development of treatments for MS. Experimental PVG.1AV1) rats of AIL-G12 subjected to PIA. autoimmune encephalomyelitis induced with myelin Linkage analysis in EAE confirmed two separate QTL oligodendrocyte glycopreotein (MOG) in rats closely overlapping with the loci controlling TNF and IL-6 mimics clinical and pathological features of human MS.19 production (Figure 1b). The first QTL, hereafter named Furthermore, the cytokine orchestration in MS and EAE Eae30, spans 6 Mb between the markers D1Rat217 and correlate well.20–22 Similarly, various animal models for D1Rat270, and showed significant linkage to all clinical RA have been used, with pristane-induced arthritis (PIA) phenotypes in addition to a linkage to the production of being the model of choice for studies on erosive RA and anti-MOG IgG2b (Supplementary Table S1; Figures 1b acute-phase responses in arthritis.23 It best fulfills the and c). Disease susceptibility and increased anti-MOG criteria for diagnosis of RA24 and is characterized IgG2b levels were conferred by the EAE-susceptible DA by pronounced bone and cartilage erosions, presence alleles. The second QTL, Eae31, covering a region of of serum rheumatoid factors and T-cell infiltrations B10 Mb between D1Rat193 and D1Rat68, was linked to in joints.25 all clinical phenotypes and to anti-MOG IgG1, IgG2b and In this study, we investigate a quantitative trait locus total IgG titers (Supplementary Table S1; Figures 1b and on rat chromosome 1, originally identified in a c). The PVG allele drove more severe disease and higher (LEW.1AV1  PVG.1AV1) F2 cross (Lewis  Piebald-Viral- levels of anti-MOG IgGs. For both QTL, there were Glaxo), which carries variants of gene(s) regulating levels effects of sex as an interactive covariate for all linked of tumor necrosis factor (TNF), interleukin (IL)-6 and IL- disease and immune sub-phenotypes in a complex 1b.26 Interestingly, the QTL overlaps loci that regulate EAE manner. On analysis of female and male rats separately and PIA.27,28 Defining genes behind this region might in Eae30, female rats displayed significant linkage to all therefore unravel genetically controlled pathways that clinical phenotypes, but not to anti-MOG IgGs, whereas regulate inflammation in general. Here we aimed first to male rats displayed significant linkage to incidence, day fine-map candidate genes responsible for the regulation of of onset and anti-MOG IgGs. For the Eae31 locus, female EAE and PIA in vivo,aswellasforin vitro cytokine rats also displayed significant linkage to all clinical production after stimulation with lipopolysaccharide phenotypes, but not to the IgG response. In males, there (LPS), and secondly to determine whether any of the was no linkage to clinical phenotypes, but instead to the human homologous genes associate with MS or ex vivo anti-MOG IgGs (Supplementary Table S1). cytokine production. We have refined this large80-Mb In an analogous linkage study on PIA, we identified QTL into two narrow loci: Eae30 and Eae31/Pia32 using the Pia32, spanning B2.1 Mb from D1Rat193 to D1Got334 tenth (G10) and twelfth (G12) generation of advanced and overlapping with Eae31 and the QTL of IL-6 and intercross line (AIL) subjected to EAE and PIA, respec- TNF. Pia32 linked to disease incidence, onset and disease tively. Subsequent investigation of candidate genes from severity (Supplementary Table S2; Figure 1d). Collec- Eae30 and Eae31 in a Nordic MS case–control cohort tively, the Eae31/Pia32 defines a narrow locus controlling demonstrated association of RGMA and IL21R with MS. two different organ-specific inflammatory diseases. Confirmation of linkage data in a congenic strain On the basis of data from the F2 cross, we developed a Results congenic rat strain, PVG.LEW-D1Rat270-D1Rat68 (here- A locus on rat chromosome 1 resolves into two independent after called PVG.LEW) by selectively breeding a frag- QTL that regulate expression of TNF and IL-6 ment from the EAE-susceptible LEW.1AV1 into a genetic A region on rat chromosome 1 was previously linked background of the major histocompatibility complex- to LPS-induced TNF responses in an F2 cross between identical, but EAE-resistant, PVG.1AV1. We used AIL to the EAE-resistant PVG.1AV1 and EAE-susceptible LE- predict how the congenic strain should behave.