Forward and Reverse Genetic Studies of AIS and Spine Development in Zebrafish

项目摘要 青少年特发性脊柱侧凸（AIS）是最常见的脊柱疾病， 在世界范围内的人群中，表现为其他方面健康的儿童，没有明显的脊椎单位结构缺陷。 AIS在年轻女性中更常见。更严重的病例需要支具或手术。尽管其医疗 重要性，对AIS的遗传基础和发病机制的理解才刚刚开始， 人类遗传研究的进展，结合斑马鱼的正向和反向遗传方法， 鼠标，我们的合作计划项目团队在第一个资助期做出了重大贡献。 在上一个资助期间，我们的项目2-斑马鱼筛选了1，673个化学诱变的F3 斑马鱼家庭的突变影响脊柱发育，产生73隐性青少年和成人脊柱侧凸 变种人对20个等位基因进行全基因组和外显子组测序，将其定位于13个染色体位点， 许多基因在正常的脊柱发育，并表明屏幕远远没有饱和。我们的前锋和 与该计划的项目1-人类和项目3-基因组学合作逆转遗传学方面的努力 识别（i）细胞外基质的组分，ii）炎症，和（ii）影响细胞外基质组装的途径。 Reissner纤维是脊柱侧凸的罪魁祸首。令人惊讶的是，脊柱侧凸的表型往往是由亚型突变引起的， 在其他必需基因中，包括adamts 9和scospondin。 利用我们富有成效的向前遗传筛选的势头，在这里，我们建议扩大我们的 受精后1至3个月的形态学脊柱侧凸筛查，使我们能够找到新的基因和途径， 系统监测脊柱侧凸表型的性别分布。新的突变位点分子 全外显子组或全基因组测序将成为人类遗传学研究的候选者 在项目1-人类和项目3-基因组学的调控元件的分析。应用 高效的基因组破坏和编辑方法，我们将验证项目1确定的候选基因座 在人类AIS患者中，包括RAPGEF 3和LBX 1基因中的蛋白质改变突变。我们将定义 我们发现的斑马鱼突变体脊柱侧凸的潜在组织和分子机制， 正向和反向遗传方法，通过全面评估骨骼形态，骨 密度、炎症途径、Reissner纤维形成和维持、转录组和生理学 脊椎管的一部分我们将测试合理选择的药物抑制表型的能力。 我们在项目2-斑马鱼中的遗传努力将与项目1-人类和 项目3-该计划的基因组学组成部分，以提供第一个基因图谱，并将定义遗传 一般来说，对脊柱正常发育至关重要的途径，特别是对AIS。

Project Abstract Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting nearly 3% of pediatric population worldwide, presenting in otherwise healthy children without overt structural defects of vertebral units. AIS is more common in young women. More severe cases require bracing or surgery. Despite its medical significance, understanding of the genetic bases and pathogenesis of AIS is just beginning and is driven by advances in human genetic studies, combined with forward and reverse genetic approaches in zebrafish and mouse, to which our collaborative Program Project team significantly contributed in the first funding period. During the previous funding period, our Project 2-Zebrafish screened 1,673 chemically-mutagenized F3 zebrafish families for mutations affecting spine development, yielding 73 recessive juvenile and adult scoliosis mutants. Whole genome and exome sequencing of 20 alleles, mapped them to 13 chromosomal loci, implicating numerous genes in normal spine development and indicating the screen is far from saturation. Our forward and reverse genetic efforts in collaboration with Project 1-Human and the Project 3-Genomics of this program identify (i) components of extracellular matrix, ii) inflammation, and (ii) pathways affecting the assembly of the Reissner fiber as culprits in scoliosis. Surprisingly, scoliosis phenotypes often result from hypomorphic mutations in otherwise essential genes, including adamts9 and scospondin. Leveraging the momentum of our productive forward genetic screen, here we propose to extend our morphologic scoliosis screen from 1 to 3 months post fertilization, allowing us to find new genes and pathways and monitor systematically sex distribution of the scoliosis phenotypes. New mutant loci molecularly characterized by whole exome or whole genome sequencing will become candidates for human genetic studies in Project 1-Human and for analyses of regulatory elements in Project 3-Genomics of this program. Applying highly efficient genome disruption and editing approaches, we will validate candidate loci identified by Project 1 in human AIS patients, including protein-altering mutations in RAPGEF3 and LBX1 genes. We will define the underlying tissue, and molecular mechanisms underlying scoliosis in the zebrafish mutants identified by our forward and reverse genetic approaches, through comprehensive assessment of skeletal morphology, bone density, inflammation pathways, the Reissner fiber formation and maintenance, transcriptomes and physiology of the spinal canal. We will test the ability of rationally chosen drugs to suppress the phenotypes. Our genetic efforts in Project 2-Zebrafish will complement and synergize with the Project 1-Human and Project 3-Genomics components of this program to provide the first atlas of genes and will define genetic pathways critical for proper spine development in general and to AIS specifically.