Genetic and epigenetic mechanisms of FSHD pathogenesis

摘要 面肩肱营养不良（FSHD）是美国最常见的肌营养不良症之一。 目前，没有有效的治疗方法，致病过程仍然没有完全了解。最 例FSHD（>95%）涉及大卫星D4 Z4重复序列的单等位基因缺失， 染色体4 q的亚端粒区（FSHD 1），而其余约5%的病例显示无D4 Z4 重复收缩（FSHD 2）。SMCHD 1基因的突变与FSHD 2相关， 通过作为疾病严重程度的修饰剂而加重FSHD 1的表型。DUX 4的表达 D4 Z4重复内编码的基因与FSHD的发生密切相关。由于过度表达 DUX 4在人肌细胞和小鼠中具有细胞毒性，因此认为DUX 4诱导的细胞毒性是DUX 4在人肌细胞和小鼠中的细胞毒性的原因。 营养不良然而，只有约0.1%的患者肌肉细胞似乎表达DUX 4，并且DUX 4表达可导致患者肌肉细胞中DUX 4的表达增加。 偶尔在未受影响个体的肌肉细胞中观察到。研究FSHD并不简单， D4 Z4重复序列和一些关键DUX 4靶基因是灵长类动物特异性的。期间 在上一个资助期，我们发现DUX 4阴性患者肌细胞表现出基因改变的证据， 与对照心肌细胞不同的表达和DUX 4靶转录因子的交叉调节 来维持DUX 4基因网络。这些发现有力地证明了FSHD机制并不简单 DUX 4诱导的细胞杀伤和进一步的研究是必要的，以了解FSHD的发病机制。我们也 开发了携带D4 Z4缺失、SMCHD 1突变或两者的基因工程突变成肌细胞系， 分别模拟FSHD 1、FSHD 2和FSHD 1严重病例，并开始表征表观遗传和 在同基因背景中定义的突变的基因表达后果。本项目的具体目标 是（1）创建额外的突变体克隆，以询问FSHD突变的后果， 具有不同疾病易感性的肌肉和早期肌形成期间;（2）研究动力学和 调节DUX 4和靶基因表达，和（3）鉴定指示疾病的修饰基因 易感性和严重性。该项目的成功结果应该揭示DUX 4和靶基因 动力学及其对FSHD发病机制的贡献，并确定疾病的关键决定因素 敏感性，这可能导致识别潜在的新的治疗靶点。

Abstract Facioscapulohumeral dystrophy (FSHD) is one of the most common muscular dystrophies in the U.S. Currently, there is no effective treatment, and the pathogenic process is still not completely understood. Most cases (>95%) of FSHD involve mono-allelic deletion of macrosatellite D4Z4 repeat sequences at the subtelomeric region of chromosome 4q (FSHD1), while the remaining ~5% of cases demonstrate no D4Z4 repeat contraction (FSHD2). Mutations in the SMCHD1 genes were linked to FSHD2, and also greatly exacerbate the phenotype of FSHD1 by acting as a modifier of the disease severity. Expression of the DUX4 gene encoded within the D4Z4 repeat is critically linked to the development of FSHD. Since overexpression of DUX4 is cytotoxic in human myocytes and mice, it is thought that DUX4-induced cytotoxicity is the cause of dystrophy. However, only ~0.1% of patient muscle cells appear to express DUX4, and DUX4 expression can occasionally be observed in muscle cells from unaffected individuals. It is not straightforward to study FSHD in model organisms as D4Z4 repeats and some of the critical DUX4 target genes are primate-specific. During the previous funding period, we found evidence that DUX4-negative patient myocytes exhibit altered gene expression distinct from control myocytes and cross-regulation of DUX4 target transcription factors contributing to sustaining the DUX4 gene network. These findings strongly argue that FSHD mechanism is not simply DUX4-induced cell killing and further investigation is necessary to understand FSHD pathogenesis. We also developed genetically engineered mutant myoblast lines carrying D4Z4 deletion, SMCHD1 mutation or both to simulate FSHD1, FSHD2 and severe cases of FSHD1, respectively and began to characterize epigenetic and gene expression consequences of defined mutations in the isogenic background. Specific Aims of this project are (1) to create additional mutant clones to interrogate the consequences of FSHD mutations in different muscles with different disease susceptibility and during early myogenesis; (2) to investigate dynamics and regulation of DUX4 and target gene expression, and (3) to identify a modifier gene(s) that dictates disease susceptibility and severity. The successful outcome of the project should reveal DUX4 and target gene dynamics and their contributions to FSHD pathogenesis and identify critical determinants for the disease susceptibility, which may lead to identification of potentially new therapeutic targets.