Translational Research in the Dystrophinopathies

描述（由申请人提供）：杜氏肌营养不良症（DMD）和 贝克肌营养不良症（BMD）是毁灭性的疾病。两者都有关联 肌营养不良蛋白基因突变，一个巨大的基因，有79个外显子分布在 2.4百万碱基的基因组序列大部分基因的缺失占所有肌营养不良蛋白突变的60%左右。其余部分包括 点突变（主要是提前终止密码子突变），小缺失 导致阅读帧的移位和（小于5%）重复。 肌营养不良蛋白基因缺失测试是商业和容易获得的，但 点突变检测则不然。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 氨基糖苷类通过诱导增加肌营养不良蛋白的表达 增加阅读量。最近，我们和其他人展示了一些规则 这种效应的特异性，越来越多的数据表明， 氨基糖苷类药物治疗可能对某些患者有益。 我们已经开发出一种方法， 进行直接序列分析的整个编码区和调控区， 肌营养不良蛋白基因，大大加快了突变的特征， 非缺失型肌营养不良蛋白病患者。使用这种方法，我们建议 在一个大型的DMD和BMD队列中， 患者，从他们的标准化和彻底的表型表征， 将获得。表型/基因型信息将在试点中汇编 肌营养不良症登记数据库。表型与 特定个体突变的序列背景将产生以下假设： 在特定序列环境中氨基糖苷类诱导的通读效率， 其将在体外双荧光素酶转染测定中进行测试。这 相同的分析将用于系统地研究其它药物化合物， 其可引起提前终止密码子的通读或移码突变， 并研究其他潜在的机制，以修改内在的框架移位 和通读。最后，我们建议开发双GFP转基因小鼠， 这将允许在体内表征组织特异性变异， 氨基糖苷类诱导的通读。虽然我们不打算执行一个 氨基糖苷类药物治疗试验目前，这项拟议的研究将确定一个 可能成为未来任何试验候选人的患者队列 机构，并可能提供一个理由，表明个别化合物， 或者剂量可能需要根据所有基因中的特定序列变化来调整 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.