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1. University of Rostock:
Rheumatoid Arthritis, is a common polygenic multi-factorial chronic inflammatory disorders that involves an interaction between genetic and environmental factors. Many linkage studies in humans and in animal models of RA, in particular murine Collagen induced arthritis (CIA), have consistently shown that there are many quantitative trait loci, QTLs, with at least 40 polymorphic genes contributing to disease susceptibility. However, identifying those putative susceptibility genes in the already identified QTL has so far not been feasible. This is due to the environmental effects and the complex epistatic interactions involving these genes. Additionally, identifying QTLs/genes controlling all disease aspects has not been completed. Therefore we have spent the last few years developing resources and new strategies that we think will enable us to identify new arthritis-susceptibility genes and their gene networks. The resources include basic animal models, QTL-congenic strains, advanced intercross lines, mitochondria substitution strains, as well as establishing methods of linkage analysis, In-vivo imaging, comparative genomics, genome-based mutagenesis, DNA sequencing and gene expression profiling.
Project:1 (Andreia Marques, ESR)
Identification of susceptibility genes/pathways for Collagen Induced Arthritis (CIA).
The Th1 and IgG2a anti-Collagen II antibody responses
A genome wide scan for QTLs was performed on 300 (DBA/1J X FVB/N) F2 mice , progeny of a cross between FVB/N (resistant) and DBA/1J (susceptible) strains. Both strains share the same MHC-haplotype, H2q. 130 informative microsatellite markers for those strains were tested in our lab and used for genotyping. The phenotypic parameters tested were anti-collagen antibody concentrations, onset and severity of clinical disease, Th1/Th2 shift in immune response to antigen and additional general immunological traits e.g. T-cell proliferation and T cell development. For the severity and onset traits 3 QTLs were identified. All were previously published. Five new QTLs controlling antibody levels, CD4/CD8 ratio and T-cell proliferation were identified. We are now focusing on finemapping the CIA27, a QTL controlling the Th1 and IgG2a anti-CII immune resonses as well as disease severity. Using a combination of genomic tools i.e. QTL-congenic production, an advanced intercross line, hapolytpe sharing analysis, we have fine mapped the locus down to a 2 Mb fragment containing 20 genes. Sequencing and expression profiling have reduced to 9 the number of candidate genes. We are currently screening those genes using an ex-vivo siRNA-based assays to determine the primary candidate gene(s). We plan to use transgenics and additional in-vivo, and ex-vivo methods for confirming the contribution of the gene(s) to arthritis and to dissect the pathways involved.
Project 2 (Dr. Lena Wester-Rosenlöf)
Dissecting the role of the mitochondria in autoimmunity using Mitochondrial substitution strains
In the FVBXDBA F2 mice we observed a strong maternal inheritance pattern of arthritis severity and diverse immune traits. This effect was mediated by an apparent genetic interaction between the mitochondrial genome and a locus on chr. 7. We went on to sequence the mitochondrial genomes of the FVB/N and DBA/1J, SJL, and C57Bl/10 strains and identified ATP8 as the mitochondrial gene responsible for this interaction. ATP8 encodes a subunit of complex V and the mutation affects Oxidative phosphorylation, reactive oxygen species production and apoptosis. To identify the interacting nuclear genes and further define the pathways involved we generated mitochondrial substitution strains involving the DBA, C57Bl/6, C57Bl/10, MRL, and NOD strains. Those are currently being used to understand the role of the mitochondria in different models of autoimmunity e.g. arthritis, Lupus, and diabetes. Our preliminary data shows a strong effect of the mutation on susceptibility to almost all common autoimmune disease.
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