Identification of genetic and molecular pathways in congenital rare disorders affecting the brain and muscle

摘要 先天性肌营养不良症（CMD）是一组异质性儿科疾病， 发育迟缓和儿童死亡率。CMD具有可变的呈现，通常影响多个 例如眼睛和大脑。虽然这种临床异质性最初阻碍了遗传分析，但ad- 下一代测序和全外显子组研究的出现大大增加了基因鉴定， 到30个基因，其中8个基因在2012-2013年鉴定，其中3个由PI鉴定。每一个新基因都提供了一个 这是一个复杂难题的不同部分，不仅为受影响的个体提供基因诊断，而且 告诉我们突变的基因如何汇聚到共享的分子途径，如蛋白质糖基化， 是的。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， 30-40%的病例仍然无法解释。与此同时，大量的已知基因阻碍了 治疗开发，因为尚不清楚如何将具有不同基因型和表型的病例分组 接受治疗 PI在过去的十年里一直专注于研究CMD的遗传学和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，我们已经开发了... 我们的中心假设，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，以及无法解释的情况下，将携带突变的新基因参与细胞， ECM相互作用或已知CMD基因中的非编码变体。拟议的研究将测试这些 通过两个独立和互补的具体目标的假设。具体目标1将利用多- 我们已经为已知的CMD基因开发了一个斑马鱼模型，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的细胞-ECM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享哪些生化缺陷，以及2）测试 糖基化的改变是否对不同形式的CMD有益。这个工作流程可以是前- 未来的治疗干预和新的疾病基因可以迅速从基因鉴定中获得， 治疗评价的重要性。具体目标2将通过测试下丘脑， 未确诊的CMD病例是由新基因的罕见突变和非 已知CMD基因的内含子/调控区中的编码突变。我们开发了下一代 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，以确定 编码，剪接和监管变异，以充分解开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， 发病机制，并为全球患者群体开发新的基因检测。

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.