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

基本信息

批准号：
10646387
负责人：
LILIANNA SOLNICAKREZEL
金额：
$ 33.96万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/10646387
关键词：
Adolescent Adopted Adult Affect Alleles Animal Model Atlases Bone Density Chemicals Child Childhood Clustered Regularly Interspaced Short Palindromic Repeats 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 Mutagens Mutation Nature Nonsense Mutation Operative Surgical Procedures Pathogenesis Pathway interactions Patients Pharmaceutical Preparations Phenotype Physiology Population Process Productivity 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 candidate validation 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 synergism transcriptome whole genome young woman

项目摘要

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凸 (AIS) 是最常见的脊柱疾病，影响近 3% 的儿童全世界范围内的人群，出现在其他方面健康的儿童中，没有明显的椎骨单位结构缺陷。 AIS 在年轻女性中更为常见。更严重的情况需要支撑或手术。尽管其医学重要的是，对 AIS 的遗传基础和发病机制的了解才刚刚开始，并且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鼠标，我们的合作项目团队在第一个资助期间做出了重大贡献。在上一个资助期间，我们的项目2-斑马鱼筛选了1,673条化学诱变的F3 斑马鱼家族的突变影响脊柱发育，产生 73 种隐性青少年和成人脊柱侧凸突变体。对 20 个等位基因进行全基因组和外显子组测序，将它们映射到 13 个染色体位点，这表明正常脊柱发育中存在大量基因，表明屏幕远未达到饱和。我们的前进和与该计划的“项目 1-人类”和“项目 3-基因组学”合作进行逆向遗传研究确定 (i) 细胞外基质的成分，ii) 炎症，以及 (ii) 影响细胞组装的途径赖斯纳纤维是脊柱侧凸的罪魁祸首。令人惊讶的是，脊柱侧凸表型通常是由亚形性突变引起的其他重要基因，包括 adamts9 和 scospondin。利用我们富有成效的前向遗传筛选的势头，我们在此建议扩大我们的研究范围受精后 1 至 3 个月进行形态学脊柱侧凸筛查，使我们能够找到新的基因和途径并系统监测脊柱侧凸表型的性别分布。新的分子突变位点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者参与项目 1-人类，并分析该项目项目 3-基因组学中的调控元件。正在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 基因的蛋白质改变突变。我们将定义我们鉴定的斑马鱼突变体的底层组织和脊柱侧弯的分子机制正向和反向遗传方法，通过骨骼形态、骨的综合评估密度、炎症途径、赖斯纳纤维的形成和维持、转录组和生理学椎管的。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-斑马鱼中的遗传工作将与项目 1-人类和项目 3 - 该计划的基因组学组成部分提供第一个基因图谱，并将定义遗传一般情况下，对于脊柱的正常发育，特别是 AIS 来说，这些通路至关重要。