面肩肱型肌营养不良症（FSHD）是最常见的成人肌营养不良症，致残率高，临床异质性大。基因定位于4q35亚端粒区，与一段大卫星序列（D4Z4 repeats）的多拷贝缺失直接相关，其上下游特异性序列SSLP和4qA/4qB具有致病选择性，但发病机制尚未阐明，推测D4Z4拷贝数减少导致内部DUX4基因异常表达，引起肌肉损害效应，但DUX4基因的具体调控途径及作用机制存在诸多矛盾，是研究的核心问题。我们前期证实中国人FSHD的致病基因型仅为SSLP161-D4Z4/PAS-4qA，支持DUX4基因的关键作用，本项目以此核心结构为切入点，采用新型基因编辑技术在细胞水平实现多靶点精准基因敲除，体外调控DUX4基因的表达，以期证实DUX4基因及侧翼序列的功能，通过BAC载体系统构建大片段插入的转基因鼠模型进行功能验证，体内调控DUX4基因表达，探索FSHD的发病机制，寻找潜在治疗靶点。

Facioscapulohumeral muscular dystrophy (FSHD) is the most common adult form of muscular dystrophy (MD), with a prevalence of approximately 1:20,000. It is an autosomal dominant disorder. The spectrum of disease severity varies widely with high disability. It results from deletion of a critical number of a repetitive 3.3-kb microsatellite sequence known as D4Z4 repeats on chromosome 4. Additionally, D4Z4 reduction is pathogenic only in a few genetic backgrounds including a special sequence of SSLP proximal to the D4Z4 repeat and the 4qA polymorphism distal to the repeat. The molecular genetic basis of FSHD is so complex that the disease mechanism has not been entirely elucidated. The inefficient repeat-mediated epigenetic repression of D4Z4 repeats is supposed to cause the variegated expression of DUX4 retrogene, encoding a double-homebox transcription factor, in skeletal muscle and result in a deregulation cascade of apoptosis, impaired muscle regeneration, and induction of an immune response. Since some disagreements regarding the details of mechanisms remain in the field, efforts are largely focusing on the dominant pathway and pathophysiological consequences of the DUX4 dysregulation. Our previous study in a large cohort of patients has confirmed that the specific sequence haplotype of SSLP161-D4Z4/PAS-4qA is almost uniquely associated with Chinese FSHD, which supports the prominent role for DUX4 gene and its polyadenylation sequence. Based on these evidences, we will utilize the new gene edit technology for multi-targeted knockout in vitro to accurately regulate the expression of DUX4 gene, which aim to identify the plausible mechanism of DUX4 regulation and explore the function of the flanking sequences of the D4Z4 repeats. Furthermore, we are going to apply BAC vector system to generate transgenic mouse model carrying large inserted DNA segments harboring the permissive haplotype SSLP161-D4Z4/PAS-4qA, which will provide a generally accepted and coherent FSHD animal model more similarly to the human genetic background. Then we are able to investigate the mechanism of DUX4 regulation in vivo and to yield new avenues for therapeutic development.