Molecular Pathphysiology of FSHD muscular dystrophy via genome-wide approaches

描述（由申请人提供）：面肩肱骨肌营养不良症（FSHD）是一种常染色体显性肌肉疾病，其特征是从面部、上臂和肩带到下肢的肌肉进行性无力和萎缩。虽然FSHD是一种转录和基因调控紊乱的共识，但导致肌肉萎缩症的分子途径和该疾病的其他独特临床特征尚不清楚。我们对代表12种神经肌肉疾病的125例肌肉活检的全基因组图谱的初步研究表明，配对样同源结构域转录因子1 （PITX1）基因在FSHD患者中特异性上调。我们在FSHD中观察到的显著PITX1过表达不可能是由于肌肉的炎症、变性/再生或其他“营养不良”变化，因为没有其他肌肉疾病（包括幼年皮肌炎、杜氏营养不良症等）出现调控。基于我们在体外和体内广泛的初步数据，我们提出了一个模型，其中PITX1在成人肌肉中过表达会调用关键的肌肉萎缩途径，并且进一步，PITX1受DUX4表达的调节。本研究的目标是进一步发展我们的病理生理模型，以显示4q35缺失、DUX4和PITX1之间的直接关系。拟议的研究在很大程度上依赖于时间序列，在体内和体外进行。基因/基因的相互作用也将被确定。在目标1中，我们建议确定Pitxl是否是DUX4的直接靶点。我们将确定在Pitxl的启动子区域一个假定的DUX4结合位点是否有功能，以及它是否被DUX4特异性和直接调控。其他DUX4目标将通过时间分析确定。将确定DUX4与潜在靶基因之间的相互作用。在目标2中，我们建议生成并表征条件肌肉特异性Pitxl转基因小鼠模型。表型将评估各种临床，功能，生化，分子和组织学参数的变化。还将评估成肌细胞的表型，包括外观，增殖，分化和对氧化应激的易感性。此外，我们将确定疾病表型是否可逆。在目标3中，我们将使用Pitxl转基因小鼠定义Pitxl表达下游的分子转录途径。我们将进行时间表达谱分析来构建由Pitxl调控的通路。Pitxl与潜在调控靶点之间的相互作用有待进一步研究。我们的初步数据显示，DUX4和Pitxl的疾病特异性上调以及肌肉萎缩相关基因的下游变化可能参与了FSHD的病理生理过程。这项拟议的研究将确定FSHD病理级联中的关键参与者，并确定它们之间的相互作用，这可能用于开发该疾病的治疗方法。

DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder that is characterized by the progressive weakness and wasting of the muscles from face, upper-arm and shoulder girdle to lower limb. While there is consensus that FSHD is a disorder of transcription and gene regulation, the molecular pathways leading to muscular dystrophy and other unique clinical features of the disease are far from clear. Our preliminary study of whole genome profiles of 125 muscle biopsies representing 12 neuromuscular disorders showed that paired-like homeodomain transcription factor 1 (PITX1) gene was specifically up-regulated in FSHD patients. The significant PITX1 over-expression we observed in FSHD can not be due to inflammation, degeneration/ regeneration, or other "dystrophic" changes in muscle, as no other muscle disease (including juvenile dermatomyositis, Duchenne dystrophy, and others) showed up- regulation. Based on our extensive preliminary data both in vitro and in vivo, we present a model where over-expression of PITX1 in adult muscle invokes key muscle atrophy pathways, and, further, that PITX1 is regulated by DUX4 expression. The goal of this current proposal is to further develop our pathophysiological model to show direct relationships between 4q35 deletions, DUX4, and PITX1. The proposed research relies heavily on temporal series, conducted both in vivo and in vitro. Gene/gene interactions will also be determined. In aim 1, we propose to determine if Pitxl is a direct target of DUX4. We will determine whether a putative DUX4 binding site in the promoter region of Pitxl is functional, and whether it is specifically and directly regulated by DUX4. Additional DUX4 targets will be identified by temporal profiling. Interaction between DUX4 and potential target genes will be determined. In aim 2, we propose to generate and characterize a conditional muscle-specific Pitxl transgenic mouse model. The phenotype will be evaluated for changes in various clinical, functional, biochemical, molecular, and histological parameters. The phenotype of myoblasts, including appearance, proliferation, differentiation and susceptibility to oxidative stress, will also be evaluated. In addition, we will determine whether the disease phenotype is reversible. In aim 3, we will define molecular transcriptional pathways downstream of Pitxl expression using the Pitxl transgenic mouse. Temporal expression profiling will be performed to construct the pathways regulated by Pitxl. Interactions between Pitxl and potential regulatory targets of Pitxl will be further studied. Our preliminary data showed that disease-specific up-regulation of DUX4 and Pitxl and downstream changes of genes involved in muscle wasting might be involved in the pathophysiology of FSHD. The proposed research will identify key players in the pathological cascades of FSHD and define the interactions among them, which could potentially be used for developing treatments of the disease.