Translational Research in the Dystrophinopathies

描述(申请人提供)：杜氏肌营养不良症(DMD)和 贝克氏肌营养不良症(BMD)是一种破坏性疾病。两者都是关联的 随着营养不良蛋白基因的突变，一个有79个外显子的巨大基因传播开来 240万个碱基的基因组序列。该基因的大部分缺失约占所有营养不良蛋白突变的60%。其余部分包括 点突变(主要是过早的终止密码子突变)，小的缺失 从而导致阅读框的移位和(少于5%的)重复。 Dstrophin基因缺失检测是商业化的，而且很容易获得，但 点突变检测则不是。DMD模型--MDX小鼠的最新研究 由于过早的终止密码子突变，已经证明了 氨基糖苷类药物诱导增加抗肌营养不良蛋白的表达 增加了通读率。最近，我们和其他人展示了一些规则 对于这种效应的特殊性，越来越多的数据表明 氨基糖苷类药物治疗对某些患者可能是有益的。 我们已经开发出了快速、稳健和经济的方法 对整个编码区和调控区进行直接序列分析 Dystrophin基因，极大地加快了突变的表征 非缺失型营养不良症患者。使用这种方法，我们建议 描述导致DMD和BMD的突变在一大群 患者，从他们标准化和彻底的表型特征， 将会被获得。表型/基因型信息将在试点中汇编 营养不良症登记数据库。表型与表型的相关性 特定个体突变的序列背景将产生以下假设 氨基糖苷在特定序列上下文中诱导通读效率， 这将在体外双荧光素酶转基因试验中进行测试。这 同样的测试将用于系统地研究其他药物化合物， 这可能导致过早终止密码子或移码突变的通读， 并研究用于修改固有帧移位的其他潜在机制 和通读。最后，我们建议开发一种双GFP转基因小鼠， 这将允许在体内表征组织特异性变异 氨基糖苷类药物诱导的通读。尽管我们不打算执行 目前氨基糖苷类药物的治疗试验，这项拟议的研究将确定一种 可能在这里或在其他地方进行任何未来试验的患者队列 机构，并可能提供一个理由，认为个别化合物 或者剂量可能需要根据所有特定的序列变化量身定制 未来的审判。

DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) are devastating disorders. Both are associated with mutations in the dystrophin gene, a huge gene with 79 exons spread over 2.4 million bases of genomic sequence. Deletions of large portions of the gene account for around 60% of all dystrophin mutations. The remainder consist of point mutations (primarily premature stop codon mutations), small deletions resulting in shift of the reading frame, and (in less than 5%) duplications. Dystrophin gene deletion testing is commercially and readily available, but point mutation testing is not. Recent studies in the mdx mouse, a model for DMD due to a premature stop codon mutation, have demonstrated the ability of aminoglycosides to increase the expression of dystrophin protein via induction of increased read-through. Recently, we and others have demonstrated some rules for the specificity of this effect, and a growing body of data suggests that aminoglycoside therapy may prove beneficial in some patients. We have developed the methodology to rapidly, robustly, and economically perform direct sequence analysis of the entire coding and regulatory regions of the dystrophin gene, greatly expediting the characterization of mutations in non-deleted dystrophinopathy patients. Using this methodology, we propose to characterize the mutations responsible for DMD and BMD in a large cohort of patients, from whom a standardized and thorough phenotypic characterization, will be obtained. Phenotype/genotype information will be compiled in a pilot dystrophinopathy registry database. Correlation of the phenotype to the sequence context of specific individual mutations will generate hypotheses of aminoglycoside-induced read-through efficiency in specific sequence contexts, which will be tested in an in vitro dual-luciferase transfection assay. This same assay will be used to systematically study other pharmaceutical compounds, which may cause read-through of premature stop codon or frameshift mutations, and to study other potential mechanisms for modifying intrinsic frame shifting and read-through. Finally, we propose to develop a dual-GFP transgenic mouse, which will allow in vivo characterization of tissue-specific variation in aminoglycoside-induced read-through. Although we do not propose to perform an aminoglycoside treatment trial at present, this proposed study will identify a cohort of patients who may be candidates for any future trials here or at other institutions, and may provide a rationale to suggest that individual compounds or dosages may need to be tailored to specific sequence variations in all future trials.