Clonal isogenic and immortalized FSHD myoblasts with or without D4Z4 contraction

描述（由申请人提供）：面肩肱骨肌营养不良症（FSHD）是一种成人常见的肌病，大多数患者是由染色体4q亚端粒D4Z4重复部分缺失引起的。FSHD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阻碍了对其病理生理的阐明。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的能力，这些模型忠实地代表了该疾病。利用在FSHD中观察到的高频率体细胞嵌合体，以及人类成肌细胞永生化的最新进展，我们的目标是产生永生化的克隆等基因肌系，这些克隆等基因肌系的区别仅在于存在或不存在突变。更具体地说，从我们已经确定的5例马赛克FSHD患者中，我们将产生5对克隆，一组有突变，一组没有突变。这些细胞系将在遗传（D4Z4重复阵列构成）、表观遗传（D4Z4的染色质结构）、转录（FSHD候选基因的表达，最明显的是DUX4）、形态学（液泡或坏死表型的存在）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。这些等基因克隆的主要优势是能够进行配对比较，仅识别FSHD特异性差异，而不存在FSHD突变。等基因克隆的这一特征将极大地促进我们通过深度转录组测序产生FSHD分子特征的最终目标。我们假设它们的不朽性、同源性和肌原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，长期目标是产生一个忠实的肌源性细胞模型，可以应用于FSHD的高通量小分子筛选。

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.