Identification of Genetic and Molecular Pathways in Congenital Rare Disorders Affecting the Brain and Muscle

摘要 先天性肌营养不良(CMD)是一组导致运动障碍的异质性儿科疾病 和发育迟缓，以及儿童死亡率。CMD具有可变的呈现方式，通常会影响多个 器官，如眼睛和大脑。虽然这种临床异质性最初阻碍了遗传分析，但更重要的是-- 下一代测序和整个外显子组研究的成功极大地增加了基因识别 到30个基因，其中8个基因是在2012-2013年间确定的，其中3个基因是PI发现的。每一个新基因都提供了一种 复杂拼图的不同部分，不仅为受影响的个体提供基因诊断，而且还 告诉我们突变的基因如何汇聚到共享的分子途径上，如蛋白质糖基- 提顿。然而，我们的团队和其他人发现，每个基因的突变只存在于一小部分 病例，30%-40%的病例仍未得到解释。与此同时，大量已知的基因阻碍了 治疗进展，因为尚不清楚如何将不同基因和表型的病例分组 接受治疗。 在过去的十年里，PI专注于研究CMD的遗传学，并开发斑马鱼模型来定义 致病突变如何影响肌肉和大脑发育。通过这些研究，我们有了发展- 揭示了我们的中心假设，即大多数CMD基因调节与细胞外基质(ECM)的相互作用 通过糖基化，而不明原因的病例要么携带涉及细胞的新基因突变- 已知的CMD基因中的ECM相互作用或非编码变体。拟议的研究将对这些进行测试 假设通过两个独立和互补的具体目标。特定目标1将利用多个- 我们已经为已知的CMD基因开发了PLE斑马鱼模型，以测试增加糖基化是否会 在不同的CMD遗传模型中，恢复肌肉和大脑中细胞-细胞外基质的相互作用。我们将1)定义 肌肉细胞和神经元中的不同基因突变共有哪些生化缺陷，以及2)测试 糖基化改变在不同形式的CMD中是否有益。然后，可以对此工作流程执行 未来的治疗干预措施和新的疾病基因可以迅速从基因识别中提取- 对治疗效果进行评价。特定目标2将通过测试假设-缩小在CMD基因发现方面的差距- SIS认为，未诊断的CMD病例是由新基因的罕见突变和非 编码已知CMD基因内含子/调节区的突变。我们已经开发出了下一代 测序和生物信息学管道将整合来自外显子组、基因组和转录组的数据，以识别- 使编码、剪接和调节变体倾斜，以充分揭示CMD的遗传学。 这些研究将通过发现不同的疾病基因如何影响CMD领域来直接影响 并通过为全球患者群体开发新的基因测试来实现这一目标。

ABSTRACT Congenital muscular dystrophies (CMDs) are a group of heterogeneous pediatric disorders leading to motor and developmental delay, and childhood mortality. CMDs have variable presentation often affecting multiple organs, such as the eyes and brain. While this clinical heterogeneity initially hindered genetic analyses, the ad- vent of next-generation sequencing and whole exome studies greatly increased gene identification reaching up to 30 genes, with eight genes identified in 2012-2013, three of which by the PI. Each new gene has provided a different piece of a complex puzzle, not only providing a genetic diagnosis for the affected individuals, but also informing us on how the mutated genes converge onto shared molecular pathways such as protein glycosyla- tion. Yet, our group and others have found that mutations in each gene are only present in a small portion of cases, and 30-40% of cases remain unexplained. In parallel, the large number of known genes has hindered therapy development as it remains unclear how cases with different genotypes and phenotypes can be grouped for treatment. The PI has focused the past decade on studying the genetics of CMD and developing zebrafish models to define how disease-causing mutations affect muscle and brain development. Through these studies, we have devel- oped our central hypotheses that most CMD genes regulate interactions with the extracellular matrix (ECM) through glycosylation, and that unexplained cases will either carry mutations in novel genes involved in cell- ECM interaction or noncoding variants in already known CMD genes. The proposed studies will test these hypotheses through two independent and complementary Specific Aims. Specific Aim 1 will leverage the multi- ple zebrafish models we have developed for known CMD genes to test whether increasing glycosylation will restore cell-ECM interactions in the muscle and brain in different genetic models of CMD. We will 1) define which biochemical deficits are shared by different genetic mutations in muscle cells and neurons, and 2) test whether changes in glycosylation can be beneficial in different forms of CMD. This workflow can then be ex- panded to future therapeutic interventions and novel disease genes can be rapidly taken from gene identifica- tion to therapy evaluation. Specific Aim 2 will close the gap in gene discovery in CMDs by testing the hypothe- sis that undiagnosed CMD cases are caused by a combination of rare mutations in novel genes and non- coding mutations in intronic/regulatory regions of known CMD genes. We have developed a next generation sequencing and bioinformatic pipeline that will integrate data from exome, genome, and transcriptome to iden- tify coding, splicing, and regulatory variants to fully unravel the genetics of CMD. These studies will directly impact the CMD fields by both discovering how different disease genes contribute to pathogenesis and by developing novel genetic tests for the global patient population.