Cofilin-2: Molecular Function and it's Role in Myopathies

描述(由申请人提供)：该项目的长期目标是了解骨钙素-2(CFL2)在骨骼肌中的作用及其在先天性肌病(CMS)中的突变。CMS的特征是相对非进行性的骨骼肌无力，早期出现低张力。线状肌病(NM)是最常见的类型，是一种遗传性疾病，肌肉中存在线状小体，构成细丝的五个不同基因发生突变。在最近的一项突破中，我发现了第六个基因CFL2在NM中发生了突变。两个临床诊断为典型NM和非特异性先天性肌病的兄弟姐妹，父母近亲出生，携带纯合子错义突变C.103G&gt；A(A35T)，并表现出肌肉中cofilin-2蛋白水平的急剧降低。与正常对照组相比，CFL2mRNA的量增加了约8倍。Cofilin-2属于AC类蛋白，包括Cofilin-1、Cofilin-2和肌动蛋白解聚因子。这些蛋白质通过引起肌动蛋白的断裂和解聚来发挥作用，在广泛的细胞功能中是必不可少的，包括运动、胞质分裂、内吞和肌肉发育。这是第一例人类AC组基因突变的确诊病例。该突变引起NM/先天性肌病的分子机制尚需了解。此外，Cofilin-2在骨骼肌中的功能还需要阐明。在目标1中，我们将筛查所有NM/其他CM病例的CFL2突变，并表征与这些突变相关的转录图谱，以 了解下游影响。在目标2中，我们将评估已识别突变的生化和功能后果。在目标3中，将使用shRNA技术下调C2C12细胞中CFL2的表达，并将评估其功能后果。在Aim 4中，将产生常规的Cfl2基因敲除和C.103G；A突变敲除小鼠模型。这个项目将向我介绍各种技术，包括微阵列分析、基因沉默和创建基因敲除/敲除小鼠模型。它将让我有机会在一个智力刺激的环境中工作，在那里我将以新颖的方式处理科学问题，促进我向独立的过渡。我的赞助人贝格斯博士、合作者/顾问达拉斯博士、多米策博士、科汉博士、昆克尔博士和麦克弗博士，以及我的新生儿科主任库伦巴纳斯博士将在这一过程中帮助我。

DESCRIPTION (provided by applicant): The long-term goals of this project are to understand the role of Cofilin-2 (CFL2) in skeletal muscle and its mutation in congenital myopathies (CMs). CMs are characterized by relatively non-progressive skeletal muscle weakness, hypotonia presenting in early life. Nemaline Myopathy (NM), the most common type, is a genetic disorder with nemaline bodies present in the muscle and mutations identified in five different genes that constitute the thin filament. In a recent breakthrough, I have identified a sixth gene, CFL2, mutated in NM. Two siblings with clinical diagnosis of typical NM and non-specific congenital myopathy born to consanguineous parents carry a homozygous missense mutation c.103G>A (A35T), and exhibit drastically reduced levels of cofilin-2 protein in their muscles. In contrast, the amount of CFL2 mRNA is increased about 8 fold compared to normal controls. Cofilin-2 belongs to AC group of proteins that include cofilin-1, cofilin-2 and actin depolymerization factor. These proteins function by causing actin severance and depolymerization, and are essential in a wide range of cellular functions including locomotion, cytokinesis, endocytosis, and muscle development. This is the first identified case of a human mutation in the AC group of genes. The molecular mechanism of NM/congenital myopathy caused by this mutation needs to be understood. In addition, cofilin-2 function in skeletal muscle needs to be elucidated. In Aim 1, we will screen all NM/other CM cases for CFL2 mutations and characterize the transcriptional profile associated with these mutations to understand downstream effects. In Aim 2, we will evaluate the biochemical and functional consequences of the identified mutation. In Aim 3, CFL2 expression in C2C12 cells will be knocked down using shRNA techniques and functional consequences will be evaluated. In Aim 4, conventional Cfl2 knockout and c.103G>A mutation knockin mouse models will be generated. This project will introduce me to a variety of techniques including microarray analysis, gene-silencing and creating knockout/knockin mouse models. It willl give me the opportunity to work in an intellectually stimulating environment, where I will approach scientific questions in novel ways fostering my transition to independence. My sponsor Dr Beggs, collaborators/advisors Dr Darras, Dr Dormitzer, Dr Kohane, Dr Kunkel and Dr Maciver, and my Neonatology Division Chief Dr Kourembanas will help me in this process.