Project II - Modeling meningomyelocele in frog using human alleles and folic acid exposure

项目 II - 使用人类等位基因和叶酸暴露模拟青蛙的脑膜脊髓膨出

• 批准号: 10300071
• 负责人: Christopher Robert Kintner
• 金额: $ 29.65万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-01 至 2025-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10300071
• 关键词: 22q11; 22q11.2; Address; Alleles; Animals; Benign; Biological Assay; Biological Models; Birth; CISH gene; Candidate Disease Gene; Cells; Chromatin; Chromosome Deletion; Clinical; Collaborations; Complex; Congenital Abnormality; Coupled; DNA Methylation; Data; Development; DiGeorge Syndrome; Disease; Dose; Effectiveness; Embryo; Environmental Risk Factor; Etiology; Event; Experimental Models; Folic Acid; Folic Acid Deficiency; Foundations; Gene Deletion; Gene Expression; Genes; Genetic; Goals; Human; Incidence; Individual; Injections; Knock-out; Lead; Literature; Mammals; Measures; Meningomyelocele; Messenger RNA; Modeling; Molecular; Morphogenesis; Movement; Mus; Mutagenesis; Mutate; Mutation; Nature; Neural Tube Closure; Neural Tube Defects; Neural Tube Development; Neural tube; Pathway interactions; Patients; Pharmacology; Phenotype; Play; Process; Rana; Recurrence; Risk; Risk Factors; Role; Spinal; Study models; Sum; Syndrome; Testing; Tetrapoda; Variant; Work; Xenopus; Xenopus laevis; base; de novo mutation; experimental study; gene environment interaction; gene interaction; genetic risk factor; genetic testing; genetic variant; in vivo; loss of function mutation; mouse model; mutant; next generation sequencing; null mutation; planar cell polarity; programs; transcriptome sequencing; xenopus genome

Abstract – Project II: Modeling meningomyelocele in frog using human alleles and FA exposure Neural tube defects (NTDs) are a relatively common birth defect with a complex etiology and gene-environment interactions (GXEs). Genetic factors play an important role, and these gene variants likely interact with one another in gene-gene interactions (GXGs). A major focus of our Program Project is the assessment of de novo mutations that can be assessed in patients with NTDs, and their modulation by environmental risk factors, such as folic acid (FA) accessibility. This complex etiology has made NTDs an extremely challenging syndrome to predict based on genetic testing and to treat clinically. This proposal is a part of a comprehensive Program Project to investigate the genetic basis of the NTD subtype known as meningomyelocele (MM), localized to the spinal neural tube, and occuring in approximately one in every 2,500 births. Project I in the application uses next generation sequencing to identify de novo gene variants that are associated with human MM patients, and assess for recurrence. Even for recurrent mutations, it is critical to functionally evaluate causality in an in vivo setting. This project exploits Xenopus as a high throughput and high content tetrapod experimental model to assess these variants, taking advantage of the fact that the morphogenetic process of neural tube formation and the underlying molecular pathways involved in neurulation are conserved between Xenopus and mammalian embryos. CrispR mutagenesis in F0 Xenopus embryos will be used to test whether genes that lie within deletions of LCR22C-D in the 22q11.2 interval and that substantially increase risk of MM in humans, cause NTDs as null mutations. Gene variants detected in the planar cell polarity pathway that may increase the risk of MM in humans will be tested using rescue experiments in Xenopus, taking advantage of quantitative assays for measuring planar axis formation. Finally, the assessment of MM gene variants will also exploit recent studies in Xenopus, indicating that folate deficiency also causes NTDs as in mammals. Interactions between genetic risk factors and folate deficiency on the incidence of NTDs will be rapidly assessed using Xenopus, as part of the overall goal to determine whether FA alters gene expression and thus the expressivity of critical gene mutants. In sum, project II will use Xenopus as model to prioritize MM gene variants identified in Project I that can be further pursued in experiments in the mouse embryo in project III. Aim 1. Test genes in the minimal 22q11.2 deletion interval for a role in NTDs using Xenopus. Aim 2. Test human MM alleles for impact on neural tube formation in Xenopus embryos. Aim 3. Test GXE by assessing the impact of FA on MM gene phenotypes in Xenopus.

摘要 - 项目II：使用人类等位基因和FA暴露在青蛙中对脑膜纤维膨胀进行建模 神经管缺陷（NTD）是一个相对常见的先天缺陷，具有复杂的病因和基因环境 相互作用（GXES）。遗传因素起着重要的作用，这些基因变异可能与一种相互作用 基因 - 基因相互作用（GXG）的另一个。我们计划项目的主要重点是从头评估 可以在NTD患者中评估的突变及其受环境风险因素的调节，例如 作为叶酸（FA）的可及性。这种复杂的病因使NTDS成为极其挑战的综合症 根据基因检测和临床治疗进行预测。该建议是综合计划的一部分 项目研究NTD亚型的遗传基础，称为脑膜瘤静脉膨胀（MM） 脊柱神经管，每2500个出生中大约有一个。应用程序I在应用程序中使用下一个 生成测序以识别与人类MM患者相关的从头基因变异的生成测序，并且 评估复发。即使对于复发性突变，在体内的功能上的因果关系至关重要 环境。该项目将Xenopus视为高吞吐量和高含量的四足动物实验模型 评估这些变体，利用神经管形成的形态发生过程和 与神经化有关的基本分子途径在爪蟾和哺乳动物之间是保守的 胚胎。 F0 Xenopus胚胎中的CRISPR诱变将用于测试缺失内的基因是否存在 在22q11.2间隔中的LCR22C-D的lcr22c-d，并且大大增加了人类MM的风险，导致NTD为null 突变。在平面细胞极性途径中检测到的基因变体可能会增加MM的风险 人类将利用定量测定法对人类进行测试 测量平面轴的形成。最后，对MM基因变体的评估还将探索最新的研究 爪蟾表明叶酸缺乏也会引起NTD，如哺乳动物。遗传风险之间的相互作用 作为NTD事件的因素和叶酸缺乏，将使用Xenopus迅速评估，作为该事件的一部分 确定FA是否改变基因表达以及关键基因突变体的表达性的总体目标。 总而言之，项目II将使用xenopus作为模型来优先考虑项目I中确定的MM基因变体 在项目III中的小鼠胚胎中进一步追求。 AIM 1。在最小22q11.2缺失间隔中使用Xenopus在NTD中扮演的测试基因。 AIM 2。测试人类MM等位基因对爪蟾胚胎中神经管形成的影响。 AIM 3。通过评估FA对MM基因表型在Xenopus中的影响来测试GXE。