article nature genetics • volume 33 • january 2003 25 Positional identification of Ncf1 as a gene that regulates arthritis severity in rats Peter Olofsson 1 , Jens Holmberg 1 , Jesper Tordsson 1 , Shemin Lu 1 , Bo Åkerström 2 & Rikard Holmdahl 1 Published online 2 December 2002; doi:10.1038/ng1058 The identification of genes underlying quantitative-trait loci (QTL) for complex diseases, such as rheumatoid arthri- tis, is a challenging and difficult task for the human genome project. Through positional cloning of the Pia4 QTL in rats, we found that a naturally occurring polymorphism of Ncf1 (encoding neutrophil cytosolic factor 1, a component of the NADPH oxidase complex) regulates arthritis severity. The disease-related allele of Ncf1 has reduced oxidative burst response and promotes activation of arthritogenic T cells. Pharmacological treatment with substances that acti- vate the NADPH oxidase complex is shown to ameliorate arthritis. Hence, Ncf1 is associated with a new autoimmune mechanism leading to severe destructive arthritis, notably similar to rheumatoid arthritis in humans. 1 Section for Medical Inflammation Research and 2 Section for Molecular Pathogenesis, Department of Cell and Molecular Biology, Sölvegatan 19, I11 BMC, Lund University, S-22184 Lund, Sweden. Correspondence should be addressed to R.H. (e-mail: rikard.holmdahl@inflam.lu.se). Introduction Rheumatoid arthritis is a chronic inflammatory disease with a prevalence of approximately 1% that primarily affects peripheral joints, where synovial inflammation leads to cartilage destruction, bone erosion and ultimately joint deformity and loss of joint func- tion. The inheritance of rheumatoid arthritis is polygenic and influenced by environmental factors. A recent investigation of published linkage analyses of rheumatoid arthritis estimated its heritable component at 60% (ref. 1). Despite large efforts, it has been difficult to identify genes and even significant loci outside the major histocompatibility complex 2,3 that regulate rheumatoid arthritis. Therefore, linkage analysis using relevant animal models represents an alternative approach to study this kind of complex disease. Rat models that meet the criteria for diagnosis of rheuma- toid arthritis in humans 4 include collagen-induced arthritis 5 and pristane (2,6,10,14-tetramethylpentadecane)-induced arthritis (PIA; ref. 6). The development of PIA in the highly susceptible DA rat is characterized by a sudden onset of arthritis two weeks after an intradermal injection of pristane followed by a chronic relaps- ing disease course with an erosive and symmetric destruction of peripheral joints and production of rheumatoid factors. We have previously reported genetic segregation analyses of arthritis-susceptible DA and arthritis-resistant E3 rats showing linkage between arthritis and several different chromosomal regions 7–9 . Most notably, different gene regions controlled differ- ent phases of the disease, such as the onset, the severity during the acute phase and the severity of destruction in the chronic relapsing phase. In addition, we found that most of the loci that we identified were shared with other arthritis models and also with a chronic relapsing model of multiple sclerosis 10 . One of these quantitative-trait loci (QTL), denoted Pia4 (pristane- induced arthritis QTL), was found to be associated with arthritis severity and joint erosions during the entire disease course. In other strains of rats, the Pia4 region has been identified in mod- els of multiple sclerosis and uveitis 10–13 , indicating that Pia4 con- trols several inflammatory diseases and contains genes with a high degree of polymorphism. Pia4 may also correspond to the homologous locus on mouse chromosome 5 (Bbaa2) that was identified using a model of Borrelia-induced Lyme disease 14 . Here, we used positional cloning to identify the gene in the Pia4 locus that is associated with arthritis severity. The Pia4 congenic fragment was limited to 300 kb, and cDNA sequencing in combi- nation with assays of function identified Ncf1 as the gene in the Pia4 region associated with the arthritis phenotype. Ncf1, also known as p47phox (phagocyte oxidase, 47 kDa) is a component of the NADPH (nicotinamide adenine dinucleotide phosphate) oxi- dase complex. The polymorphic Ncf1 allele in DA rats led to dif- ferences in enzyme activity rather than to quantitative differences in expression, and caused a lower oxygen burst that resulted in severe arthritis, presumably owing to activation of arthritogenic T cells in lymphoid organs. We also show that activators of the NADPH oxidase complex can prevent arthritis onset. These data suggest a new pathway, involving the NADPH oxidase complex, that can result in severe inflammation in autoimmune diseases. Results Congenic rats are protected against arthritis The Pia4 QTL on rat chromosome 12 has been shown to be inherited in an additive fashion, as DA rats carry the disease- promoting allele of Pia4 in the original F2 intercross between E3 and DA rats 7 . To isolate the genetic fragment harboring the Pia4 locus, we transferred a 20-cM fragment of the resistant E3 allele onto the DA background, producing a DA.pia4 congenic strain. This congenic strain was backcrossed with DA parental rats for 12 generations to avoid interference from other E3 contaminat- ing loci. To ensure that all phenotypic differences originated exclusively from the genetic difference in the Pia4 region, we carried out all arthritis experiments using DA littermate con- trols originating from F2 intercrosses between heterozygous DA.pia4a/b (a = E3, b = DA) rats. © 2003 Nature Publishing Group http://www.nature.com/naturegenetics