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

斑马鱼 AIS 和脊柱发育的正向和反向遗传学研究

• 批准号: 10458401
• 负责人: LILIANNA SOLNICAKREZEL
• 金额: $ 35.7万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10458401
• 关键词: Adolescent; Adopted; Adult; Affect; Alleles; Animal Model; Atlases; Bone Density; CRISPR/Cas technology; Chemicals; Child; Childhood; Collaborations; Complement; Critical Pathways; Databases; Defect; Deformity; Development; Diagnosis; Engineering; Essential Genes; Ethylnitrosourea; Extracellular Matrix; Family; Fertilization; Fiber; Funding; Genes; Genetic; Genetic Screening; Genetic study; Genome; Genome engineering; Genomics; Goals; Grant; Human; Human Genetics; Idiopathic scoliosis; Induced Mutation; Inflammation; Maintenance; Maps; Mediating; Medical; Missense Mutation; Modeling; Modernization; Molecular; Molecular Analysis; Monitor; Morphogenesis; Morphology; Mus; Muscle; Mutation; Nature; Nonsense Mutation; Operative Surgical Procedures; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Physiology; Population; Process; Proteins; Regulatory Element; Research; SOX6 gene; Sex Distribution; Spinal Canal; Spinal Diseases; Structural defect; Testing; Tissue Banks; Tissues; Transgenic Organisms; Variant; Vertebral column; Zebrafish; base; exome; exome sequencing; experience; genetic approach; genome editing; genome sequencing; human disease; innovation; loss of function; mutant; neuroinflammation; nonsynonymous mutation; null mutation; prevent; prime editing; programs; pulmonary function; reverse genetics; scoliosis; screening; skeletal; spine bone structure; transcriptome; whole genome; young woman

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.

项目摘要 青少年特发性脊柱侧弯（AIS）是最常见的脊柱疾病，影响了近3％的小儿 在全球范围内的人口，在没有明显的椎骨结构缺陷的情况下出现在其他健康的儿童中。 AI在年轻女性中更为普遍。更严重的病例需要支撑或手术。尽管有医疗 意义，了解AIS的遗传碱和发病机理才刚刚开始，并由 人类遗传研究的进步，以及斑马鱼和反向遗传方法的结合 鼠标，我们的协作计划项目团队在第一个资金期间为其做出了重大贡献。 在上一个资金期间，我们的2-泽布拉夫筛选了1,673个化学量化F3 斑马鱼家庭用于影响脊柱发育的突变，产生73个隐性少年和成人脊柱侧弯 突变体。整个基因组和外显子组测序的20个等位基因，将它们映射到13个染色体基因座，与 正常脊柱发育中的许多基因并表明筛网远非饱和。我们的前锋和 反向遗传努力与项目1-Human和该计划的第三基因组合作 识别（i）细胞外基质的组成部分，ii）炎症，以及（ii）影响组装的途径 Reissner纤维作为脊柱侧弯的罪魁祸首。令人惊讶的是，脊柱侧弯表型通常是由于肌莫尔型突变而引起的 在其他必要基因中，包括ADAMTS9和Scospondin。 利用我们有效的前向遗传筛选的势头，我们在这里提议扩展我们 受精后1到3个月的形态脊柱侧弯筛查，使我们能够找到新的基因和途径 并监测脊柱侧弯表型的系统性别分布。新的突变基因座分子 以整个外显子组或整个基因组测序的特征将成为人类遗传研究的候选者 在项目1-Human和该计划项目3基因组学中的监管元素中进行分析。申请 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Rapgef3和LBX1基因中的蛋白质改变突变。我们将定义 我们的潜在组织和分子机制在我们 通过骨骼形态的全面评估，骨骼形态，骨骼的前进和反向遗传方法 密度，炎症途径，Reissner纤维形成和维护，转录组和生理学 脊柱管。我们将测试合理选择的药物抑制表型的能力。 我们在第2泽布拉夫项目中的遗传努力将与该项目1-Human和 该程序的项目3基因组成分提供基因的第一个地图集，并将定义遗传 一般和AIS的适当脊柱发育至关重要的途径。