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，影响近3%的儿童 世界各地的人口，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。 人工授精在年轻女性中更为常见。更严重的病例需要支撑或手术。尽管它的医疗保健 对AIS的意义、遗传基础和发病机制的了解才刚刚开始，是由 人类遗传学研究的进展，结合斑马鱼的正向和反向遗传方法 在第一个资助期，我们的协作计划项目团队对此做出了重大贡献。 在之前的资助期间，我们的项目2-斑马鱼筛选了1,673个化学诱变的F3 斑马鱼家族突变影响脊柱发育，导致73例隐性幼年和成年脊柱侧弯 变种人。20个等位基因的全基因组和外显子组测序，将它们映射到13个染色体座位，暗示 在正常的脊椎发育中有大量的基因，这表明屏幕远未饱和。我们的前锋和 与项目1-人类和项目3--本计划的基因组学合作进行的反向遗传努力 确定(I)细胞外基质的成分，ii)炎症，和(Ii)影响 Reissner纤维是脊柱侧弯的罪魁祸首。令人惊讶的是，脊柱侧弯的表型通常是由亚形突变引起的。 在其他必需基因中，包括adamts9和scospondin。 利用我们富有成效的前向基因筛查的势头，在这里，我们建议延长我们的 在受精后1到3个月进行形态脊柱侧弯筛查，使我们能够发现新的基因和途径 并系统监测脊柱侧弯表型的性别分布。新的分子突变基因座 以全外显子组或全基因组测序为特征的将成为人类遗传学研究的候选 在项目1-人类和项目3-基因组学中用于分析本计划的调控元件。施药 高效的基因组中断和编辑方法，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RAPGEF3和LBX1基因的蛋白质改变突变。我们将定义 我们发现的斑马鱼突变体脊柱侧弯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、骨骼 密度、炎症途径、Reissner纤维的形成和维持、转录本和生理学 在椎管内。我们将测试合理选择药物抑制表型的能力。 我们在项目2-斑马鱼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提供了第一份基因图谱，并将定义基因 对脊柱正常发育至关重要的通路，特别是对AIS来说。

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.