Project 1. Gene Variants and Their Interactions Defining Human NTD Risk

项目 1. 定义人类 NTD 风险的基因变异及其相互作用

基本信息

批准号：
9528613
负责人：
MARGARET ELIZABETH ROSS
金额：
$ 57.7万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/9528613
关键词：
ARHGEF10 gene Address Affect Alleles Anencephaly and spina bifida X linked Animal Model Basic Science Binding Binding Sites Biochemical Biological Biological Assay CRISPR/Cas technology Candidate Disease Gene Cell Line Cell Polarity Cell model Cells Cellular Morphology Child Clinical Research Code Collaborations Complex Cyst DNA Resequencing DNA sequencing Data Data Set Databases Detection Disease Dose Embryo Engineering Enhancers Environmental Exposure Epithelial Folic Acid Funding Gene Transfer Generations Genes Genetic Genetic Diseases Genetic Enhancement Genetic Predisposition to Disease Genome Genomic DNA Genomics Goals Grant Human Hydrogen Peroxide In Vitro Individual Infant Inherited Intervention Metabolism Methodology Methods Modeling Molecular Mus Mutagenesis Mutation Neural Tube Closure Neural Tube Defects Neural tube Neuroepithelial Cells Nitrates Nitrogen Nucleic Acid Regulatory Sequences Nutrient Oxidative Stress Oxygen Parents Pathway interactions Patients Peroxonitrite Pluripotent Stem Cells Population Prevalence Prevention Protein Chemistry Proteins Protocols documentation Qatar Regimen Risk Sampling Shotguns Single Nucleotide Polymorphism Spinal Dysraphism Superoxides System Technology Testing Tight Junctions Untranslated RNA Variant Work base blastomere structure candidate selection clinical practice cohort exome falls gene interaction genetic variant genome editing high throughput screening human embryonic stem cell line human subject in vivo insertion/deletion mutation medical schools mouse model mutant next generation nitration nitrosative stress novel occludin prevent programs rare variant relating to nervous system risk variant transcription factor transcriptomics whole genome

项目摘要

ABSTRACT PROJECT 1: Gene Variants and their Interactions Defining Human NTD Risk Neural tube defects (NTDs), primarily spina bifida and anencephaly, arise from a complex interplay of multiple gene interactions and environmental exposures. After 30 years of clinical and basic research, the field remains unable to accurately predict the risk for an individual couple of having a child affected by NTD, how folic acid (FA) works to prevent NTDs, whether or what dose of FA is likely provide effective prevention for them, or whether there is another nutrient/supplement or intervention that would provide greater benefit. The recent confluence of advances in genomics and computational genetics, enhanced by information from genetic mouse models, capabilities of molecular biological and biochemical detection in embryonic systems, now provide outstanding opportunity to address this complex genetic disorder. In the initial funding period, Project 1 has accumulated 200 whole genome sequences (WGS) from cases and 200 controls and has identified rare nonsense, frameshift and non-coding variants associated with spina bifida. This has thus far generated over 100 candidate gene and transcription factor binding sites associated in both expected and novel molecular interaction pathways in humans that are enriched for rare sequence variants in NTD cases compared to non-malformed controls and public databases. Among the most striking findings are that pathways modulating or affected by oxidative stress are heavily represented among the rare variants in our NTD cohort. In the renewal, we will employ a powerful high throughput method using molecular inversion probes (MIPs) to resequence a replication cohort of over 2,000 NTD cases and validate the most highly significant genes and non-coding, regulatory regions of the genome associated with NTD risk. Cutting edge CRISPR-Cas9 dependent genome editing in hESCs and mice will probe the functional impact of identified variants on neuroepithelial cell polarity, proliferation, differentiation, survival and the generation of reactive oxidative/nitrosative species (RONS). With Projects 2 and 3 we will explore the impact of these variants on oxidative stress in in vitro hESC models bearing patient variants, and the ability of compounds that manipulate RONS in these mutant cells to exacerbate or reverse aberrant cell morphology, differentiation and, in genome edited mouse models, promote successful neural tube closure (NTC). Building upon the results obtained in our original funding period, Project 1, in collaboration with Projects 2 and 3, rigorously applies next generation DNA sequencing, genome editing, animal modeling, protein chemistry, and transcriptomic approaches to illuminate the causes of human NTDs, with a goal to ultimately reduce their prevalence.

摘要项目 1：定义人类 NTD 风险的基因变异及其相互作用神经管缺陷 (NTD)，主要是脊柱裂和无脑畸形，是由以下因素的复杂相互作用引起的：多基因相互作用和环境暴露。经过30年的临床和基础研究，该领域仍然无法准确预测一对夫妇生育受 NTD 影响的孩子的风险，叶酸 (FA) 如何预防 NTD，叶酸是否或什么剂量可能提供有效的预防对他们来说，或者是否有其他营养素/补充剂或干预措施可以提供更大的益处。基因组学和计算遗传学的最新进展的融合，通过来自以下信息的增强：遗传小鼠模型、胚胎系统中分子生物学和生化检测的能力、现在为解决这种复杂的遗传性疾病提供了绝佳的机会。在最初的资助期间，项目1已经积累了200个全基因组序列（WGS）病例和 200 个对照，并已识别出与脊柱裂。迄今为止已经生成了 100 多个候选基因和转录因子结合位点与人类预期的和新颖的分子相互作用途径相关，这些途径富含稀有物质与非畸形对照和公共数据库相比，NTD 病例中的序列变异。其中最引人注目的发现是，氧化应激调节或影响的途径在很大程度上具有代表性这是我们 NTD 队列中的罕见变异之一。在更新中，我们将采用强大的高通量方法使用分子倒置探针 (MIP) 对 2,000 多个 NTD 病例的复制队列进行重新测序，验证与相关的基因组中最重要的基因和非编码调节区域被忽视的热带病风险。 hESC 和小鼠中最先进的 CRISPR-Cas9 依赖性基因组编辑将探索已识别的变异对神经上皮细胞极性、增殖、分化、存活和功能的影响反应性氧化/亚硝化物质（RONS）的产生。通过项目 2 和 3，我们将探索这些变异对携带患者变异的体外 hESC 模型中氧化应激的影响，以及操纵这些突变细胞中的RONS以加剧或逆转异常细胞形态的化合物，分化，并在基因组编辑的小鼠模型中促进成功的神经管闭合（NTC）。基于我们最初资助期间获得的成果，项目 1 与项目2和3，严格应用下一代DNA测序、基因组编辑、动物建模、蛋白质化学和转录组学方法阐明人类 NTD 的原因，目标是最终降低其患病率。