Genetic and epigenetic mechanisms of FSHD pathogenesis

摘要 面肩肱骨营养不良(FSHD)是美国最常见的肌营养不良症之一。 目前，尚无有效的治疗方法，致病过程尚不完全清楚。多数 FSHD病例(&gt；95%)涉及大卫星D4Z4重复序列的单等位基因缺失 染色体4Q亚端粒区(FSHD1)，其余~5%的病例未发现D4Z4 重复收缩(FSHD2)。Smchd1基因的突变与FSHD2有关，而且在很大程度上 通过作为疾病严重程度的修饰物来加重FSHD1的表型。DUX4的表达 D4Z4重复序列中编码的基因与FSHD的发生密切相关。由于过度表达 DUX4在人和小鼠心肌细胞中具有细胞毒性，目前认为DUX4诱导的细胞毒性是导致DUX4致病的原因 营养不良。然而，只有~0.1%的患者肌肉细胞似乎表达DUX4，并且DUX4的表达可以 偶尔在未受影响的个体的肌肉细胞中观察到。研究FSHD并非易事。 模式生物如D4Z4重复和一些关键的DUX4靶基因是灵长类特有的。在.期间 在之前的资助期，我们发现了DUX4阴性患者心肌细胞表现出基因改变的证据 不同于对照心肌细胞的表达及DUX4靶转录因子的交叉调节 来维持DUX4基因网络。这些发现有力地证明了FSHD机制并不是简单的 DUX4诱导的细胞杀伤和进一步的研究对于了解FSHD的发病机制是必要的。我们也 开发的携带D4Z4缺失和/或Smchd1突变的基因工程突变成肌细胞系 分别模拟FSHD1、FSHD2和FSHD1重症病例，并开始表征表观遗传学和 等位基因背景中特定突变的基因表达后果。本项目的具体目标 是(1)创建额外的突变克隆，以询问FSHD突变在不同的 不同疾病易感性和早期肌肉发生的肌肉；(2)研究动力学和 DUX4和靶基因表达的调节，以及(3)鉴定决定疾病的修饰基因(S) 易感性和严重性。项目的成功结果应该揭示DUX4和靶基因 动力学及其在FSHD发病机制中的作用和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.