Pristane-induced arthritis (PIA) in rats is a model mimicking RA. Arthritis is induced by a subcutaneous injection of the adjuvant oil pristane at the base of the tail and signs of arthritis appear after about 14 days. The subsequent development of arthritis is chronic and relapsing in the susceptible DA strain, with an erosive and symmetric destruction of peripheral joints and the disease fulfils the classical RA criteria [16,17]. PIA is T cell-dependent and there is no evidence for an influence of B cells or arthritogenic antibodies in contrast to CIA, the primarily used arthritis model in mice, in which arthritogenic antibodies to CII play a significant role .
To identify the genes contributing to the disease, the arthritis-susceptible DA strain was crossed with the arthritis-resistant E3 strain [19,20]. In a number of both intercrosses and backcrosses involving more than 1,000 rats, the major quantitative trait loci (QTLs) could be identified (Fig. 3). These loci could clearly be separated to control specific phases of the disease course, i.e. onset, severity, and chronicity (Fig. 4). One of the QTLs, Pia4 located on chromosome 12, was found to be associated with severity of PIA. By insertion of the genetic fragment of interest from the resistant strain (in this case E3) into the genome of the susceptible DA strain through conventional backcross breeding (more than ten generations), we created a congenic strain. At this stage, the congenic fragment was 20 cM and we found that the congenic rat developed a milder arthritis than the parental DA strain. In fact, the difference was significant using fewer than ten rats in each group, which is a criterion for going further to clone the underlying gene/s. In other words, the prerequisite
was that we had transformed the trait to be more Mendelian. On this basis, we started a process of collecting and testing small cohorts of animals having recombinations within this fragment in order to establish subcongenic strains . Each new subcongenic strain was screened for the presence of the disease-associated genes by testing them for PIA susceptibility. With this method, the fragment could be reduced to contain only two genes, Ncfl and Gtf2i. No differential expression of either Ncf1 or Gtf2i could be detected and only Ncfl (alias p47phox) had polymorphism alterations in the coding sequence leading to changes in amino acid sequence in the translated protein, suggesting that Ncf1 was essential for arthritis susceptibility. One of the alterations was at position 106 ATG/GTG and resulted in a Met/Val alteration, the other one was at position 153 ATG/ACG and resulted in a Met/Thr alteration. Brown Norway (BN) rats, which are resistant to arthritis, share all genotypes but one with DA, the alteration in position 153, suggesting that this alteration is important for the Ncf1 function. Consequently, this approach allowed us to
QTLs controlling specific subtraits of arthritis in E3xDA rat crosses
Pia4, PiaS, Pia9, PiulO, Pia!2, J Pia!J, Pia 14. Pial5
identify a causative gene, which could now be subjected for further functional analysis and studies of this new pathogenic pathway associated with arthritis severity.
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