The Role of Germline Mutations in the Ras/MAPK Pathway on Skeletal Myogenesis

描述（由申请人提供）：骨骼肌发生是一个动态过程，在这个过程中，肌肉前体细胞首先增殖，然后融合形成多核肌管，最终成熟为骨骼肌纤维。Ras/丝裂原活化蛋白激酶（MAPK）通路是一种被充分研究的癌症通路，在肌发生调控中起着关键作用，特别是在成肌细胞增殖向分化的转变过程中。早期研究表明，高水平的Ras/MAPK通路激活会破坏早期肌肉形成。然而，对于Ras及其下游效应级联如何调节和影响发育过程中肌发生的重要步骤，我们的理解存在重大空白。“RASopathies”是一组新定义的医学遗传综合征，是已知的最大的多先天性异常综合征群之一，每1000人中就有1人以上受到影响。由编码Ras/MAPK通路组分的各种关键基因的种系突变引起的ras病具有先天性张力低下或肌肉无力的共同表型特征。Costello综合征（CS）和心-面-皮（CFC）综合征是两种肌肉表型最严重的rasopathy。我们最近在CS和CFC患者中发现了一种新的肌病，定义为非神经功能障碍的肌肉内在异常。另外，我们初步

DESCRIPTION (provided by applicant): Skeletal myogenesis is a dynamic process in which muscle precursor cells first proliferate and then fuse to form multinucleated myotubes that ultimately mature into skeletal muscle fibers. The Ras/Mitogen-activated protein kinase (MAPK) pathway, a well-studied cancer pathway, plays a critical role in the regulation of myogenesis, particularly during the switch from myoblast proliferation to differentiation. Early studies have demonstrated that high levels of Ras/MAPK pathway activation disrupt early myogenesis. However, there are critical gaps in our understanding as to how Ras and its downstream effector cascades regulate and affect vital steps in myogenesis during development. The "RASopathies", a newly defined group of medical genetic syndromes, are one of the largest groups of multiple congenital anomaly syndromes known, affecting more than 1 in 1000 individuals. Caused by germline mutations in various key genes encoding components of the Ras/MAPK pathway, the RASopathies share a common phenotypic feature of congenital hypotonia, or weak muscles. Costello syndrome (CS) and cardio-facio-cutaneous (CFC) syndrome are two RASopathies with the most severe muscle phenotype. We have recently identified the presence of a novel myopathy, defined as an intrinsic abnormality of muscle that is not attributable to nerve dysfunction, in individuals with CS and CFC. In addition, our preliminary studies provide support for our hypothesis that dysregulation of Ras/MAPK signaling disrupts both early myogenesis by inhibiting myoblast differentiation, and later stages of muscle development following differentiation, by inhibiting muscle growth. The goal of the proposed research is to understand how myogenesis is affected by Ras/MAPK dysregulation, as well as the specific mechanism of action underlying this effect. We will examine novel germline mutations identified in the RASopathies to help us understand how Ras dysregulation affects muscle development. Our Specific Aims are designed to determine 1) how skeletal muscle is disrupted by dysregulation of Ras/MAPK pathway signaling in CS and CFC; 2) the mechanisms by which Ras/MAPK dysregulation causes disruption of skeletal muscle myogenesis, and 3) if small molecule inhibitors and small interfering RNAs (siRNA) can reverse the effects of dysregulated Ras/MAPK signaling during myogenesis. We will use mouse models of CS and CFC to elucidate how skeletal muscle is disrupted by distinguishing what aspect of the myopathy is due to inhibition of myoblast differentiation and what is due to muscle fiber formation from post-differentiation inhibition of muscle growth. We will elucidate the specific mechanisms by which Ras/MAPK signal dysregulation inhibits myogenesis using primary myoblasts derived from CS and CFC mouse models. Results derived from those experiments will be used to evaluate the effectiveness of rationally chosen inhibitors to correct the developmental effects of a dysregulated Ras pathway using in vitro and in vivo models of myogenesis.