Single-nucleus profiling of FSHD heterogeneity

面肩肱营养不良（FSHD）是最常见的肌营养不良症之一。大多数 例与染色体4 q上的D4 Z4重复序列（FSHD 1）缩短有关， SMCHD 1转录抑制基因的突变与一小部分FSHD患者有关 （FSHD 2）。SMCHD 1中的突变也极大地加剧了FSHD 1的表型，从而作为SMCHD 1的修饰剂。 FSHD 1中疾病的严重程度。D4 Z4重复序列中DUX 4基因的异常表达是 与FSHD 1和FSHD 2的发育有关。然而，只有一小部分病人的肌肉 细胞表达DUX 4蛋白，偶尔也可以在未受影响的肌肉细胞中观察到。 个体这表明DUX 4单独表达可能不足以促进FSHD的发展。因此，在本发明中， DUX 4基因在患者肌肉细胞中是如何上调的，以及它是如何导致FSHD的。 发病机制有待进一步研究。由于D4 Z4重复序列不存在于小鼠基因组中， 患者肌肉细胞对于评估FSHD特异性细胞变化是必需的。然而，高变异性 在仅具有表达DUX 4的细胞的小子集的样品中， 辛普森悖论（Simpson's Paradox）虽然单细胞转录组分析可以解决这个问题， 然而，“单细胞捕获”对于大的多核肌管不是最佳的。因此，我们建议 进行单核测序，以确定细胞异质性的程度，并测试是否存在小的 细胞群携带疾病特征并驱动FSHD发病机制。在目标1中，我们计划执行 单核RNA测序，以确定对照、FSHD 1和 FSHD 2成肌细胞分化前后。在目标2中，我们将重点分析DUX 4- 在FSHD 1和FSHD 2中表达和不表达细胞，以确定DUX 4依赖性和非依赖性 两种FSHD的变化。因此，本研究的目的是确定DUX 4介导的致病性 患者肌细胞的变化，并了解患者细胞群的异质性程度， 捕获可能的“致病”细胞。该项目将直接影响我们对 FSHD生物学和新型诊断/治疗策略的潜在开发以及患者的优化 在乎

Facioscapulohumeral dystrophy (FSHD) is one of the most prevalent muscular dystrophies. The majority of cases are associated with shortening of the D4Z4 repeat sequences on chromosome 4q (FSHD1) while mutations in the SMCHD1 transcriptional repressor gene were linked to a smaller subset of FSHD patients (FSHD2). Mutations in SMCHD1 also greatly exacerbate the phenotype of FSHD1, thus acting as a modifier of the disorder's severity in FSHD1. Abnormal expression of the DUX4 gene present in the D4Z4 repeats is linked to the development of both FSHD1 and FSHD2. However, only a small percentage of patient muscle cells express DUX4 protein, which can also occasionally be observed in muscle cells from unaffected individuals. This suggests that DUX4 expression alone may not be sufficient for FSHD development. Thus, exactly how the DUX4 gene is upregulated in patient muscle cells and how it contributes to FSHD pathogenesis need to be further investigated. Since D4Z4 repeats are not present in the mouse genome, patient muscle cells are essential for assessing FSHD-specific cellular changes. However, high variability among samples with only a small subset of cells expressing DUX4 may exacerbate the averaging artifact of population analysis (Simpson's paradox). Though single-cell transcriptome analysis should address this conundrum, “single-cell capture” is not optimal for large multinucleated myotubes. Thus, we propose to perform single-nucleus sequencing to determine the extent of cellular heterogeneity and test if a small population of cells carries the disease signature and drives FSHD pathogenesis. In Aim 1, we plan to perform single-nucleus RNA-sequencing to determine the extent of transcriptome heterogeneity in control, FSHD1 and FSHD2 myoblasts before and after differentiation. In Aim 2, we will focus on comparative analysis of DUX4- expressing and non-expressing cells in FSHD1 and FSHD2 to determine DUX4-dependent and –independent changes in two types of FSHD. The goal of this study, therefore, is to define DUX4-mediated pathogenic changes in patient muscle cells and to understand the extent of heterogeneity of patient cell population and capture the possible “disease-driving” cells. The project should have direct impact on our understanding of FSHD biology and potential development of novel diagnostic/therapeutic strategies and optimization of patient care.