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

摘要-项目II：使用人类等位基因和FA暴露在青蛙中建模脑膜脊髓膨出 神经管缺陷是一种相对常见的出生缺陷，其病因和基因环境复杂 交互作用（GXE）。遗传因素起着重要的作用，这些基因变异可能与一个基因相互作用。 另一个是基因-基因相互作用（GXGs）。我们的计划项目的一个主要重点是重新评估 可以在NTD患者中评估的突变，以及它们受环境风险因素的调节， 叶酸（FA）的可及性。这种复杂的病因使得NTD成为一种极具挑战性的综合征， 根据基因检测进行预测并进行临床治疗。该提案是一项全面计划的一部分， 研究NTD亚型（称为脑膜脊髓膨出（MM））的遗传基础的项目， 脊髓神经管，大约每2,500个新生儿中就有一个。应用程序中的项目I使用next 世代测序以鉴定与人类MM患者相关的从头基因变异，和 评估复发情况。即使对于复发性突变，在体内试验中功能性评估因果关系也是至关重要的。 设置.该项目利用非洲爪蟾作为高通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 在非洲爪蟾和哺乳动物之间，神经形成的潜在分子途径是保守的 胚胎F0非洲爪蟾胚胎中的CrispR诱变将用于测试是否存在缺失的基因 LCR 22 C-D在22q11.2间隔中的表达，并显著增加人类MM的风险，导致NTD为空 突变。在平面细胞极性通路中检测到的可能增加MM风险的基因变异 人类将使用非洲爪蟾的救援实验进行测试，利用定量分析， 测量平面轴形成。最后，MM基因变异的评估也将利用最近的研究， 非洲爪蟾，表明叶酸缺乏也会导致NTD，如哺乳动物。遗传风险之间的相互作用 因素和叶酸缺乏对NTD发病率的影响将使用非洲爪蟾进行快速评估，作为 总体目标是确定FA是否改变基因表达，从而改变关键基因突变体的表达能力。 总之，项目II将使用非洲爪蟾作为模型，优先考虑项目I中鉴定的MM基因变异， 在项目III的小鼠胚胎实验中进一步进行。 目标1。使用非洲爪蟾测试最小22q11.2缺失间隔中的基因在NTD中的作用。 目标2.测试人类MM等位基因对非洲爪蟾胚胎神经管形成的影响。 目标3：通过评估FA对非洲爪蟾MM基因表型的影响来测试GXE。

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.