IDENTIFICATION OF GENES REGULATING ARTHRITIS IN RATS

胶原性关节炎是一种成熟的关节炎实验动物模型，已被用来深入了解类风湿性关节炎的发病机制。这项建议的总体目标是确定调控这种实验性关节炎发展的非MHC基因。在导致提出这一建议的实验中，利用了这样一个事实，即拥有相同MHC单倍型RT1av1的两个近交系大鼠对同源鼠II型胶原诱导的关节炎的敏感性显著不同，尽管这两个品系都表现出胶原自身抗体的产生。对关节炎易感性DA和关节炎抗性ACI大鼠的F2杂交进行全基因组扫描，在大鼠2号染色体上发现了一个非MHC DA起源的数量性状基因座(QTL)Cia7，在2号和10号染色体上还发现了另外两个提示基因座。这三个基因座映射到基因组区间，该区间与先前在小鼠和人类基因组中发现的包含与不同形式的自身免疫性疾病易感性有关的基因的区域相同，进一步强调了这项研究的相关性。这一建议假设上述三个QTL包含将胶原免疫诱导的无症状自身免疫状态转变为一种坦率的自身免疫性疾病的基因。第一个目的是构建基因引导的SPEED同源大鼠，将DA片段引入ACI背景，反之亦然。与这三个QTL的性别交互作用将在同源动物中进行研究。在后期阶段，将产生多基因大鼠来确定这三个基因座的交互作用。第二个目标是识别基因。为了实现这一目标，将使用两种基本方法。首先，将采用候选基因策略，利用小鼠和人类基因组同源图谱数据，筛选候选基因外显子突变，然后进行测序，如果适用的话。某些候选基因的差异表达将在从滑膜组织和T细胞产生的cDNA文库中进行分析。第二种策略将遵循更标准的方法，首先通过产生重组子将同源基因间隔缩小到1 cM，然后分离区域中的转录本，克隆和测序。这两种策略都将通过识别参与反应的候选替代免疫学亚型来补充，这些亚型应该提供对基因作用机制的洞察和免疫表型的替代标记，从而促进基因发现过程。

Collagen-induced arthritis is a well-established experimental animal model of arthritis that has been used to gain insight into the pathogenesis of rheumatoid arthritis. The overall objective of this proposal is to identify the non-MHC genes that regulate development of this form of experimental arthritis. In experiments that led up to present proposal, advantage was taken of the fact that two inbred rat strains sharing the same MHC haplotype, RT1av1, differed strikingly in their susceptibility to arthritis induced with homologous rat type II collagen, although both strains exhibited the production of collagen autoantibodies. A genome-wide scan in a F2 cross between arthritis-susceptible DA and arthritis-resistant ACI rats identified one non-MHC DA- originated quantitative trait locus (QTL), Cia7, on rat chromosome 2, and two other suggestive loci on chromosome 2 and 10. A significant sex effect was also identified. These three loci map to genomic intervals syntenic to regions previously identified in the mouse and human genomes containing genes involved in the susceptibility to different forms of autoimmune diseases, further emphasizing the relevance of this study. This proposal hypothesizes that the three QTL described above contain genes that act to convert the state of asymptomatic autoimmunity induced by the collagen immunization into a frank autoimmune disease. The first aim is construction of genotype-guided speed congenic rats, introducing DA segments into ACI background, and vice-versa. Sex interaction with the three QTL will be studied in the congenic animals. At later stages, polycongenic rats will be generated to determine the interaction of the three loci. The second aim is identification of the genes. In order to accomplish this goal, two basic approaches will be used. First, a candidate gene strategy will be employed, taking advantage of mouse and human genome homology mapping data, and screening candidate gene exons for mutations, followed by sequencing, when applicable. Differential expression of certain candidate genes will be analyzed in cDNA libraries generated from synovial tissue and T cells. The second strategy will follow a more standard approach, first narrowing the congenic interval to 1 cM through generation of recombinants, followed by isolation of transcripts in the regions, cloning and sequencing. Both of these strategies will be supplemented by identification of candidate surrogate immunologic sub-phenotypes involved in the response that should provide both insight into the mechanism of gene action and alternative markers for immunologic phenotypes thus facilitating the gene discovery process.