Can Somatic Mutations Mediate Anti-La Autoimmunity

描述(由申请人提供)：临床自身免疫性疾病的机制已被证明是很难阐明的。有证据表明，分子模仿、亚显性表位、免疫忽视和细胞凋亡机制的失败在各种实验系统中都得到了令人信服的证明。或许，还有其他机制存在。我们认为，体细胞突变是启动自身免疫的一种可能机制，并进一步建议对一个针对干燥综合征和系统性红斑狼疮的核抗原La的自身免疫实例进行实验评估。据推测，体细胞突变产生的新蛋白质结构以前是免疫系统所不知道的。一旦针对新结构(S)的传出免疫反应延伸到抗原的非突变结构，那么就建立了自身免疫反应。偶然的是，我们在DNA、RNA和蛋白质水平上都检测到了抗La沉淀阳性患者的人La突变热点中的体格移位突变的例子。最近的初步数据表明，这种突变可能发生在多达50%的La阳性患者中，而在健康献血者和年龄、性别和种族匹配的对照组中没有检测到突变。在热点插入或缺失会导致随后的氨基酸序列改变，从而截断La抗原。这种突变可能会产生新的B细胞和T细胞表位。事实上，我们在自身免疫患者中发现了针对热点区域的抗体。此外，我们创建了基因组突变和天然La基因构建物，并建立了转基因动物，这些构建物在其中永久表达。这些基因组结构将被用来创建基因组克隆，使我们能够开发出一种对本地人La耐受的动物，在这种动物中，我们可以打开人类突变La形式的表达。在此，我们提出了一个实验计划，以进一步探索La的体细胞突变及其产生自身免疫的潜力。我们已经开发出高灵敏度和特异性的检测方法，以检测DNA、RNA和蛋白质水平的体细胞突变。根据功率计算，我们将通过将这些技术应用于抗La阳性和抗La阴性的年龄、性别和种族匹配的患者以及健康对照来扩展我们的初步研究。此外，我们将确定La的体细胞突变是否可以在实验动物和自身免疫患者中产生新的B细胞和T细胞表位。最后，使用Cre loxP系统，我们将生成一个动物模型，模拟在人类中观察到的特定体细胞突变。这些实验应该有助于确定体细胞突变是否是一种可信的自身免疫机制。

DESCRIPTION (provided by applicant): The mechanisms of clinical autoimmune disease have proven to be difficult to elucidate. Evidence implicating molecular mimicry, sub-dominant epitopes, immune ignorance, and failure of the apoptotic cellular apparatus as contributing to the mechanisms of autoimmunity have been convincingly demonstrated in various experimental systems. Perhaps, other mechanisms exist. We propose that somatic mutation is a possible mechanism to initiate autoimmunity and further propose to experimentally evaluate an example of autoimmunity to the nuclear antigen La, which is targeted in Sjogren's syndrome and systemic lupus erythematosus. Presumably, the new protein structures created by somatic mutation would have been previously unknown to the immune system. Once the efferent immune response against the new structure(s) extends to non-mutated structures of the antigen, then an autoimmune response would be established. Serendipitously, we detected examples of somatic frame shift mutations in a mutational hot spot of human La in anti-La precipitin-positive patients at both the DNA, RNA and protein level. Recent preliminary data indicate that such mutations may occur in up to 50% of La positive patients, while no mutations were detectable in healthy donors and age-, sex-, and race-matched controls. Insert or deletion in the hot spot leads to a subsequently altered amino acid sequence with truncation of the La antigen. Such mutations could result in novel B- and T-cell epitopes. Indeed, we found antibodies to the hot spot region in autoimmune patients. Furthermore, we created genomic mutant and native La gene constructs and established transgenic animals in which these constructs were permanently expressed. These genomic constructs will be used to create a genomic clone allowing us to develop an animal tolerant to native human La in which we can switch on the expression of the human mutant La form. We herein present an experimental plan to further explore somatic mutation of La and its potential for generating autoimmunity. We have developed highly sensitive and specific assays to detect the somatic mutation at the DNA, RNA and protein level. According to power calculations we will extend our preliminary studies by applying these techniques to anti-La positive and anti-La negative age-, sex- and race-matched patients as well as healthy controls. Furthermore, we will determine whether or not somatic mutation in La can create novel B- and T-cell epitopes in experimental animals and autoimmune patients. Finally, using the Cre loxP system we will generate an animal model imitating the particular somatic mutations observed in man. These experiments should help establish whether somatic mutation is a plausible mechanism for autoimmunity.