MOLECULAR MECHANISMS OF THE MYOTONIC DYSTROPHY MUTATION

强直性肌营养不良症(DM)是成人中最常见的遗传性神经肌肉疾病，全球发病率为1/8000。DM突变是编码丝氨酸-苏氨酸蛋白激酶(DMPK)基因的3‘非翻译区(3’UTR)上一个不稳定的CTG三联体重复序列的扩张。然而，它导致疾病的机制尚不清楚。我们和其他人已经发现，突变的DMPK mRNA被困在DM细胞的细胞核内，并形成明显的、稳定的mRNA焦点。此外，我们还证明了突变的DMPK 3‘UTRmRNAs对基因表达有显著的负面影响。此外，我们还发现，突变的DMPK 3‘UTRmRNA在成肌细胞中的表达导致成肌细胞融合和分化的缺陷，表明该RNA反式作用于其他转录本的表达，并导致与疾病相关的细胞表型。这项研究旨在通过解决DM是RNA介导的疾病过程的范例这一假设来理解DM的分子生物学。拟议的实验将旨在评估和确定DMPK信使RNA(MRNA)对基因表达的影响。将首先从细胞水平研究正常和突变的DMPK 3‘UTRmRNA的影响，然后从生化角度研究，最后通过建立转基因小鼠模型在体内进行研究。本研究提出的假设是：强直性肌营养不良是一种由突变的DMPK mRNA介导的RNA代谢失调与DM的病理生理有关的疾病。本研究的具体目的是：1)研究DM突变对细胞培养模型的影响；2)鉴定突变的DMPK 3‘UTRmRNA表达改变的基因；3)建立小鼠模型，研究DM突变对体内RNA代谢的影响及其在DM发病机制中的作用。这一建议的长期目标是了解DM突变发挥作用的分子机制，以便深入了解DM的病理生理学，允许开发合适的动物模型，并最终导致更合理的DM治疗干预方法。

Myotonic dystrophy (DM) is the most common inherited neuromuscular disorder in adults with a global incidence of 1 per 8000. The DM mutation was found to be an expansion of an unstable CTG triplet repeat in the 3' untranslated region (3'UTR) of a gene encoding a serine-threonine protein kinase (DMPK). However, the mechanism by which it causes disease is unknown. We and others have found that the mutant DMPK mRNA is trapped within the nucleus of DM cells and forms distinct, stable foci of mRNA. In addition, we have demonstrated that the mutant DMPK 3'UTR mRNAhas significant negative effects on gene expression. Furthermore, we have identified that expression of the mutant DMPK 3'UTR mRNA in myoblasts causes defects in myoblast fusion and differentiation, demonstrating that this RNA work in trans on the expression of other transcripts, and causes a disease relevant cellular phenotype. This study is directed at understanding the molecular biology of DM by addressing the hypothesis that DM is a paradigm for RNA mediated disease processes. The proposed experiments will be aimed at assessing and determining the effect of the DMPK messenger RNA (mRNA) on gene expression. The effects of the normal and mutant DMPK 3'UTR mRNA will be studied initially at the cellular level, secondly from a biochemical persepective and finally in vivo through the creation of a transgenic murine model. The hypothesis to be tested by this proposal is that: Myotonic dystrophy is a disease in which dysregulation of RNA metabolism mediated by the mutant DMPK mRNA contributes to the pathophysiology of DM. The specific aims of this proposal are directed at: 1) studying the effects of the DM mutation in a cell culture model, 2) identifying genes whose expression is altered by the presence of the mutant DMPK 3'UTR mRNA and 3) the establishment of a murine model to study the in vivo effects of the DM mutation on RNA metabolism and their contribution to DM pathogenesis. The long term objectives of this proposal are to understand the molecular mechanisms by which the DM mutation functions in order to provide insight into the pathophysiology of DM, to allow for the development of appropriate animal models, and to eventually lead to a more rational approach to therapeutic intervention in DM.