Can Somatic Mutations Mediate Anti-La Autoimmunity

体细胞突变能否介导抗 La 自身免疫

• 批准号: 6547598
• 负责人: A Darise Farris
• 金额: $ 25.87万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6547598
• 关键词: B lymphocyte; Sjogren's syndrome; T lymphocyte; autoantibody; autoantigens; autoimmunity; clinical research; enzyme linked immunosorbent assay; gene mutation; genetically modified animals; human subject; laboratory mouse; mass spectrometry; matrix assisted laser desorption ionization; p53 gene /protein; patient oriented research; protein structure function; systemic lupus erythematosus

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.

描述（由申请人提供）：事实证明，临床自身免疫性疾病的机制很难阐明。在各种实验系统中，人们可以令人信服地证明了涉及分子模仿，亚显性表位，免疫无知和凋亡细胞设备失败的证据。也许还有其他机制。 我们建议，体细胞突变是启动自身免疫性的可能机制，并进一步提议通过实验评估对核抗原LA的自身免疫性的例子，该核抗原LA是针对Sjogren综合征和系统性狼疮的红斑狼疮。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未知的。一旦对新结构的传出免疫反应扩展到抗原的非突变结构，则将建立自身免疫反应。 偶然地，我们在DNA，RNA和蛋白质水平的抗LA沉淀蛋白阳性患者中，在人LA的突变热点中检测了体细胞移位突变的实例。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，而在健康的供体以及年龄，性别和种族匹配的对照中未检测到突变。在热点中插入或缺失会导致随后随着LA抗原的截断而改变的氨基酸序列。这种突变可能导致新型的B-和T细胞表位。确实，我们发现自身免疫性患者的热点区域的抗体。此外，我们创建了基因组突变体和本地LA基因构建体，并建立了转基因动物，其中这些构建体被永久表达。这些基因组构建体将用于创建一个基因组克隆，使我们能够开发出对本地人类LA的动物耐受性，在该动物中，我们可以开启人类突变体形式的表达。我们在这里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性的潜力。我们已经开发了高度敏感和特定的测定法，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阴性年龄，性别和种族匹配的患者以及健康对照组来扩展初步研究。此外，我们将确定LA中的体细胞突变是否可以在实验动物和自身免疫性患者中产生新颖的B和T细胞表位。最后，使用CRE LOXP系统，我们将生成一个模仿人类观察到的特定体细胞突变的动物模型。这些实验应有助于确定体细胞突变是否是自身免疫性的合理机制。