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难以阐明。在各种实验系统中，已经令人信服地证明了分子模拟、亚优势表位、免疫无知和凋亡细胞装置的失败对自身免疫机制的贡献。也许还有其他机制。 我们提出，体细胞突变是一种可能的机制，启动自身免疫，并进一步提出实验评估的核抗原La，这是针对干燥综合征和系统性红斑狼疮的自身免疫的一个例子。据推测，由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。一旦针对新结构的传出免疫应答延伸至抗原的非突变结构，则将建立自身免疫应答。 偶然地，我们在DNA、RNA和蛋白质水平上检测到抗La沉淀素阳性患者中人La突变热点的体细胞移码突变的例子。最近的初步数据表明，这种突变可能发生在高达50%的La阳性患者中，而在健康供体和年龄，性别和种族匹配的对照中没有检测到突变。热点中的插入或缺失导致随后改变的氨基酸序列和La抗原的截短。这种突变可能导致新的B-和T-细胞表位。事实上，我们在自身免疫患者中发现了热点区域的抗体。此外，我们创建了基因组突变体和天然La基因构建体，并建立了这些构建体永久表达的转基因动物。这些基因组构建体将用于创建基因组克隆，从而允许我们开发对天然人La耐受的动物，其中我们可以开启人突变La形式的表达。我们在此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。我们已经开发了高度敏感和特异性的检测方法来检测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，我们将确定是否体细胞突变的La可以创建新的B-和T-细胞表位在实验动物和自身免疫性患者。最后，使用Cre loxP系统，我们将产生一个动物模型，模仿在人类中观察到的特定体细胞突变。这些实验将有助于确定体细胞突变是否是自身免疫的一个合理机制。

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.