Molecular Pathogenesis of Myotonic Dystrophy

描述（由申请人提供）：强直性肌营养不良症（DM）是成人发病型肌营养不良症的最常见形式，影响全球1/8500人。它是显性遗传，影响多个器官系统。致病突变是在转录但非翻译的基因组区域中扩展的三（CTG）和四（CCTG）核苷酸重复。DM的主要致病机制涉及由扩增等位基因的RNA转录物表达引起的毒性RNA功能获得。本提案的目标是确定含有扩展重复序列的RNA引起进行性骨骼肌营养不良和心律失常的分子机制，这是死亡和发病的主要原因。在上一个资助期，我们证明了致病机制涉及选择性剪接的失调，我们确定了负责特定症状的靶基因。我们还发现，这些目标是由两个家族的RNA结合蛋白的拮抗性调节，这两个家族都是以前根据CUG RNA结合活性鉴定的。我们将定义的机制，扩大CUG RNA诱导发病机制，特别强调这两个蛋白质家族的个别成员的作用。首先，虽然很明显CUG重复RNA是致病性的，但致病性所需的RNA的具体形式是未知的。我们将使用一个既定的测定，以确定序列，蛋白结合，和结构特征的RNA所需的诱导剪接失调的反式。蛋白质的直接相互作用与RNA相关的异常RNA加工将被确定。第二，基因的错误调节有助于肌病和心律失常将被确定使用新的生化和减法的方法。第三，诱导表达扩增的CUG RNA的稳定细胞系将用于表征毒性RNA的分布和代谢以及对与细胞毒性相关的RNA结合蛋白表达的影响。第四，我们将开发多功能系的转基因小鼠使用Cre/LoxP策略，以诱导特定组织和早期发育中高水平的扩增CUG RNA。这些研究将提供细胞和小鼠模型，以确定改变的调控途径和受这种新的疾病机制影响的基因。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy affecting 1 in 8500 people worldwide. It is dominantly inherited and affects multiple organ systems. Causative mutations are expanded tri-(CTG) and tetra-(CCTG) nucleotide repeats in transcribed but non-translated genomic regions. A major pathogenic mechanism of DM involves a toxic RNA gain-of-function caused by expression of RNA transcripts from the expanded alleles. The goal in this proposal is to determine the molecular mechanism by which RNA containing the expanded repeats causes progressive skeletal muscle dystrophy and cardiac arrhythmias, the predominant causes of mortality and morbidity. In the previous funding period we demonstrated that the pathogenic mechanism involves misregulation of alternative splicing and we identified target genes responsible for specific symptoms. We also found, that these targets are regulated antagonistically by two families of RNA binding proteins both identified previously based on CUG RNA binding activity. We will define the mechanism by which expanded CUG RNA induces pathogenesis with specific emphasis on the roles of individual members of these two protein families. First, while it is clear that CUG repeat RNA is pathogenic, the specific form of the RNA required for pathogenicity is unknown. We will use an established assay to define the sequence, protein binding, and structural features of RNA required for induction of splice-misregulation in trans. Proteins whose direct interactions with the RNA correlate with aberrant RNA processing will be identified. Second, genes whose mis-regulation contributes to myopathy and arrhythmias will be identified using novel biochemical and subtractive approaches. Third, stable cell lines inducibly expressing expanded CUG RNA will be used to characterize the distribution and metabolism of toxic RNA and the consequences on the expression of the RNA binding proteins that are relevant to cell toxicity. Fourth, we will develop versatile lines of transgenic mice using a Cre/LoxP strategy to induce high levels of expanded CUG RNA in specific tissues and in early development. These studies will provide cellular and mouse models to define altered regulatory pathways and the genes affected by this novel disease mechanism.