Single-nucleus profiling of FSHD heterogeneity

面肩肱骨营养不良(FSHD)是最常见的肌营养不良症之一。大多数人 4例与染色体4Q上的D4Z4重复序列缩短(FSHD1)有关，而 Smchd1转录抑制基因突变与FSHD患者的一小部分有关 (FSHD2)。Smchd1的突变也极大地加剧了FSHD1的表型，从而充当了 在FSHD1中，这种疾病的严重性。D4Z4重复序列中DUX4基因的异常表达 与FSHD1和FSHD2的发展相联系。然而，只有一小部分患者的肌肉 细胞表达DUX4蛋白，这种蛋白偶尔也可以在未受影响的肌肉细胞中观察到 个人。这表明仅有DUX4的表达可能不足以促进FSHD的发生。因此， DUX4基因在患者肌肉细胞中究竟是如何上调的，以及它在FSHD中的作用 发病机制有待进一步研究。由于D4Z4重复序列不存在于小鼠基因组中， 患者肌肉细胞是评估FSHD特异性细胞变化的关键。然而，高度的可变性 在只有一小部分细胞表达DUX4的样本中，可能会加剧 人口分析(辛普森悖论)。尽管单细胞转录组分析应该解决这个问题 令人费解的是，“单细胞捕获”并不是大型多核肌管的最佳选择。因此，我们建议 进行单核测序以确定细胞异质性的程度，并测试是否有小的 细胞群携带疾病特征并驱动FSHD的发病机制。在目标1中，我们计划执行 单核RNA测序以确定对照组、FSHD1和FSHD1转录组的异质性程度 分化前后的FSHD2成肌细胞。在目标2中，我们将重点比较分析DUX4- FSHD1和FSHD2中表达和不表达细胞确定DUX4依赖和非依赖 两种类型的FSHD的变化。因此，这项研究的目标是定义DUX4介导的致病 患者肌肉细胞的变化，并了解患者细胞群体和 捕获可能导致疾病的细胞。这个项目应该直接影响我们对 FSHD生物学、新诊断/治疗策略的潜在开发和患者的优化 关心。

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.