A new genetic approach to identify the Idd9.3 type 1 diabetes susceptibility gene

项目总结 已发现30多个自身免疫性1型糖尿病(T1D)易感基因(称为IDD)在 非肥胖糖尿病(NOD)小鼠，人类疾病的自发动物模型。其中， Idd9.3基因座位于4号染色体上的1.22Mb区域，包含17个蛋白质编码和12个蛋白质编码 非编码基因，其中Tnfrsf9(编码CD137)是首选候选基因。C57BL/10(B10)-派生 Idd9.3赋予T1D抗性，而Nod派生的间隔有助于疾病的发展。由于 该区域的紧密连锁，可用于进一步剖析Idd9.3基因座的同源菌株尚未 以更准确地确定Tnfrsf9是否是潜在的基因。人类全基因组 关联研究已将TNFRSF9与几种自身免疫性疾病联系起来，CD137在功能上相互作用 在具有已知人类T1D基因(例如，TNFAIP3)的免疫途径中。因此，对其进行深入研究具有重要意义。 CD137在T1D中的作用此前有报道称，与Nod来源的CD137相比，Nod来源的CD137功能低下 B10 CD137。另一方面，我们证明了CD137缺陷的NOD小鼠，由锌指产生 核酸酶(ZFN)介导的诱变，对T1D具有抗性。我们在这里进一步展示了CD137在 CD4和CD8 T细胞分别抑制和促进NOD小鼠T1D的发育，而CD137 缺乏主要影响CD8 T细胞，导致疾病保护。因此，NOD小鼠的T1D发育 携带Tnfrsf9的不同等位基因(B10、Nod或空等位基因)受其综合效应的影响 不同的细胞类型具有不同的功能。需要更好的方法来最终确定Tnfrsf9是否是 比较CD8T细胞和CD8T细胞中NOD和B10等位基因的功能。美国人的能力 ZFN技术在缺乏生殖系可传递胚胎的小鼠品系中特异性敲除基因 干细胞还使我们能够针对B10Idd9.3区域的同源NOD小鼠靶向Tnfrsf9。我们的目标 在这方面的应用是通过建立一对Tnfrsf9是否为Idd9.3的潜在基因来最终确定 仅表达Tnfrsf9的一个亲本等位基因(NOD或B10)的遗传相同的菌株，并进一步 检测CD137的等位基因变异是否改变了其在T1D中的主导作用模式。

PROJECT SUMMARY More than 30 autoimmune type 1 diabetes (T1D) susceptibility loci (termed Idd) have been identified in the nonobese diabetic (NOD) mouse, a spontaneous animal model for the human disease. Among those, the Idd9.3 locus has been mapped to a 1.22 Mb region on Chromosome 4 containing 17 protein-coding and 12 non-coding genes, of which Tnfrsf9 (encoding CD137) is the top candidate. The C57BL/10 (B10)-derived Idd9.3 confers T1D resistance, whereas the NOD-derived interval contributes to disease development. Due to the tight linkage of the region, congenic strains that can be used to further dissect the Idd9.3 locus have not been made available to more precisely determine if Tnfrsf9 is the underlying gene. Human genome wide association studies have linked TNFRSF9 to several autoimmune diseases, and CD137 functionally interacts in immune pathways with known human T1D genes (e.g., TNFAIP3). Thus, it is important to further study the role of CD137 in T1D. It has been previously reported that NOD-derived CD137 is hypofunctional compared to B10 CD137. On the other hand, we demonstrate that CD137-deficient NOD mice, generated by zinc-finger nuclease (ZFN) mediated mutagenesis, are resistant to T1D. We further show here that CD137 expression in CD4 and CD8 T-cells respectively suppresses and promotes T1D development in NOD mice, but CD137 deficiency dominantly affects CD8 T-cells leading to disease protection. Thus, T1D development in NOD mice carrying different alleles of Tnfrsf9 (B10, NOD, or the null allele) is influenced by the combined effects of its distinct functions in different cell types. A better approach is needed to conclusively determine if Tnfrsf9 is the Idd9.3 gene and to compare the function of NOD and B10 alleles in CD4 versus CD8 T-cells. The ability of the ZFN technology to specifically knock out a gene in mouse strains lacking germ-line transmittable embryonic stem cells has also allowed us to target Tnfrsf9 in the NOD mice congenic for the B10 Idd9.3 region. Our goal in this application is to definitively determine if Tnfrsf9 is the Idd9.3 underlying gene by establishing a pair of genetically identical strains where only one parental allele of Tnfrsf9 (NOD or B10) is expressed, and to further examine whether allelic variation of CD137 changes its dominant mode of action in T1D.