DM2: Murine and cell Culture Models of CCUG RNA toxicity

描述（由申请人提供）：强直性肌营养不良（DM）是成人肌营养不良的最常见形式，可由19号染色体（DM 1）或3号染色体（DM 2/PROMM）上的突变引起。DM 1由肌强直性肌营养不良蛋白激酶基因（DMPK）的3'非翻译区和同源结构域基因SIX 5的启动子区中的CTG扩增引起。虽然DM 1突变在1992年被分离出来，但多年来一直难以理解这种不改变基因蛋白质编码部分的突变是如何导致这种显性遗传的多系统疾病的。提出的机制包括：DMPK单倍不足;包括同源结构域基因SIX 5在内的区域基因表达减少;以及RNA中CUG扩增的致病作用。小鼠模型表明，这些机制中的每一个都有助于DM 1的发病机制，DM 1可能是一种区域性基因疾病。为了阐明DM的致病机制，我们研究了第二种形式的强直性肌营养不良，其在人类中引起惊人相似的一系列临床特征，包括肌强直、肌病、虹彩性白内障、心律失常和DM特有的一组血清学变化。我们将DM 2基因座定位于染色体3q 21（Nature Genetics 19：196-198，1998），并且最近报道DM 2是由锌指蛋白9（ZNF 9）基因的内含子1中的重复扩增引起的（Science，293：864-867）。DM 1和DM 2之间的临床和分子相似性表明，在RNA而不是蛋白质水平表达的CUG和CCUG扩增本身可能是致病的，并导致两种疾病共同的多系统特征。我们建议产生转基因小鼠和细胞培养模型，以测试CCUG重复包含转录的影响，并更好地了解参与疾病过程的分子机制。我们的具体目标是：（1）建立可逆的小鼠模型，以测试致病性并表征在骨骼肌中表达的含有CCUG扩增的转录物的下游效应。(2)建立并表征复制强直性肌营养不良2型多系统特征的BAC转基因小鼠模型。(3)建立细胞培养模型，以评估CCUG长度对RNA灶形成、细胞分化和选择性剪接下游分子变化的分子效应。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on chromosome 19 (DM1) or 3 (DM2/PROMM). DM1 is caused by a CTG expansion in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK) and the promoter region of the homeodomain gene SIX5. Although the DM1 mutation was isolated in 1992, for many years it has been difficult to understand how this mutation, which does not alter the protein-coding portion of a gene, causes this dominantly-inherited multisystemic disorder. Suggested mechanisms have included: DMPK haploinsufficiency; reduced expression of regional genes including the homeodomain gene SIX5; and pathogenic effects of the CUG expansion in RNA. Mouse models have suggested that each of these mechanisms contributes to DM1 pathogenesis and that DM1 may be a regional gene disorder. To clarify the pathogenic mechanism of DM, we have studied a second form of myotonic dystrophy that causes a strikingly similar constellation of clinical features in humans including myotonia, myopathy, iridescent cataracts, cardiac arrhythmias, and the specific set of serological changes characteristic of DM. We mapped the DM2 locus to chromosome 3q21 (Nature Genetics 19:196-198, 1998) and recently reported that DM2 is caused by a repeat expansion in intron 1 of the zinc finger protein nine (ZNF9) gene (Science, 293:864-867). Clinical and molecular parallels between DM1 and DM2 indicate that CUG and CCUG expansions expressed at the RNA but not the protein level can themselves be pathogenic and cause the multisystemic features common to both diseases. We propose generating transgenic mice and cell culture models to test the effects of CCUG repeat-containing transcripts and to better understand the molecular mechanisms involved in the disease process. Our specific aims are: (1) To generate a reversible murine model to test the pathogenicity and characterize the downstream effects of transcripts containing the CCUG expansion expressed in skeletal muscle. (2) To generate and characterize a BAC transgenic mouse model that replicates the multisystemic features of myotonic dystrophy type 2. (3) To develop a cell culture model to evaluate the molecular effects of CCUG length on the formation of RNA foci, cellular differentiation and downstream molecular changes in alternative splicing.