Clonal Isogenic and Immortalized FSHD Myoblasts with or without D4Z4 Contraction

描述(申请人提供)：面肩肩周型肌营养不良症(FSHD)是一种常见的成人肌肉疾病，是由大多数患者4Q染色体亚端粒D4Z4重复序列的部分缺失引起的。FSHD目前还没有治愈或有效的治疗方法，其复杂的遗传和表观遗传病因长期以来阻碍了对其病理生理学的阐明。对FSHD疾病机制的有限了解阻碍了我们开发可靠的细胞和动物模型来忠实地代表这种疾病的能力。利用在FSHD中观察到的高频率的体细胞嵌合体，以及在人类成肌细胞永生化方面的最新进展，我们的目标是建立永生化的克隆等基因肌源性细胞系，其差异仅在于突变的存在或不存在。更具体地说，从我们已经确认的5个嵌合型FSHD患者中，我们将产生5对克隆，一组带有突变，一组没有突变。这些细胞系将在遗传(D4Z4重复序列构成)、表观遗传(D4Z4的染色质结构)、转录(FSHD候选基因的表达，尤其是DUX4)、形态(存在空泡或坏死表型)和功能水平(对氧化应激的敏感性和参与体内和体外肌肉分化的能力)上进行表征。这些同基因克隆的主要优势是，只有存在或不存在FSHD突变才有区别，能够进行配对比较，仅识别FSHD特异性差异。等基因克隆的这一特征将极大地促进我们的最终目标，即通过深层转录组测序为FSHD生成分子签名。我们推测，它们的永生化、同源性和肌源性将使它们成为理想的细胞系，以促进我们对FSHD病理生理学的理解，并结合FSHD分子特征的建立，用于高通量药物筛选。因此，我们的长期目标是建立一种可靠的肌源性细胞模型，可以应用于FSHD的高通量小分子筛查。 公共卫生相关性：该项目旨在为FSHD患者培育具有相同基因的永生化肌肉细胞系。这意味着，除了突变之外，它们在基因上是相同的。我们希望这些细胞系将有助于了解疾病的机制，并将用于治疗目的的小分子筛选。

DESCRIPTION (provided by applicant): Facioscapulohumeral muscular dystrophy (FSHD) is a common myopathy in adults and is caused by partial deletion of the D4Z4 repeat in the subtelomere of chromosome 4q of most patients. There is no cure or effective treatment for FSHD and its complex genetic and epigenetic etiology has long precluded elucidation of its pathophysiology. This limited knowledge of FSHD disease mechanism has hampered our ability to develop validated cellular and animal models faithfully representing the disease. Taking advantage of the high frequency of somatic mosaicism observed in FSHD, and recent advances in human myoblast immortalization, we aim to generate immortalized clonal isogenic myogenic cell lines that only differ by the presence or absence of the mutation. More specifically, from each of the 5 mosaic FSHD patients we have identified, we will generate 5 pairs of clones, one set with and one set without mutation. These cell lines will be characterized at the genetic (D4Z4 repeat array constitution), epigenetic (chromatin structure of D4Z4), transcriptional (expression of FSHD candidate genes, most notably DUX4), morphological (presence of a vacuolar or necrotic phenotype) and functional level (sensitivity to oxidative stress and ability to participate in muscle differentiation in vivo and in vitro). The principal advantage of these isogenic clones, differing only in the presence or absence of the FSHD mutation, is the ability to do paired comparisons that identify only FSHD-specific differences. This feature of the isogenic clones will greatly facilitate our final aim of generating a molecular signature for FSHD by deep transcriptome sequencing. We hypothesize that their immortality, isogenicity and myogenic origin will make them ideal cell lines to advance our understanding of the pathophysiology of FSHD and, in combination with the establishment of a molecular signature for FSHD, for high throughput drug screens. Therefore, the long-term goal is to generate a faithful myogenic cell model that can be applied in high throughput small molecule screens for FSHD. PUBLIC HEALTH RELEVANCE: This project aims to generate immortalized muscle cell lines of FSHD patients that are isogenic. This means that they are genetically identical except for the mutation. We expect that these cell lines will aid the understanding of the disease mechanism and will be useful for small molecule screens for therapeutic purposes.