Analysis of Poison Exon Inclusion in Genes Associated with Neurodevelopmental Disorders and Autism Spectrum Disorder

项目摘要/摘要 每一百名新生儿中就有一名受到神经发育障碍(NDDS)的影响，这可能会导致 一系列令人衰弱的症状，包括早发性癫痫发作、发育迟缓和智力残疾。 应用全外显子组/基因组测序检测新城疫 可能存在变异，但在相当大比例的病例中，遗传原因仍未确定。分析了几个问题 当编码变异不能与疾病相关时，深内含子非编码变异提供了诊断。 例如，SCN1A“20N”毒物外显子中的内含子变异会导致功能丧失，从而导致Drave. 综合症，一种在出生第一年会导致长时间癫痫发作的新城疫。毒物外显子是基因的一种机制 因包含有毒外显子而导致提前终止密码子导致的无稽之谈的规定- 介导的信使核糖核酸转录本的衰退。初步分析表明，毒素外显子包涵体(PEI)在 SCN1A和SCN8A在胚胎大脑发育过程中减少，与总RNA成反比 表情。大脑是早期神经元发育中选择性剪接的温床，因此它是 新的毒物外显子即使不是可能的，也是可信的，仍然没有特征。这一提议的总体假设 PEI是一种在神经元发育早期差异调节基因表达的机制， 而影响发育重要基因中PEI的内含子变异也会导致疾病 表型。在目标1中，钠和钙电压门控通道阿尔法亚基家族将被评估为 检测和鉴定新的毒物外显子。已知和新颖的PEI将在开发过程中确定 大脑使用公开可用的rna-seq实验。在目标2中，该分析将扩大100倍以上，以 检测和评估3000多个与NDD和自闭症谱系障碍(ASD)相关的基因的PEI 将评估大脑发育和PEI进化的驱动力。毒物外显子的比较分析 将使用k-均值层次聚类和无监督随机森林机器学习进行 确定驱动PEI利用的主要生物学因素的方法。在《目标3》中，内含子变体将是 新城疫和房间隔缺乏症先证者在已知和假想PEI区域的分析及其对这些变异的影响 将对疾病表型进行计算评估。这些实验将揭示出对 发育中大脑中的选择性剪接，并为目前未知的NDD提供潜在的遗传学解释 疾病表型。这项研究将在哈德逊·阿尔法生物技术研究所进行，该研究所允许 通过以下补充培训课程培养科学家在动态、动手的环境中磨练技能 这是必要的。除了提案中概述的研究外，培训还将包括负责任的行为课程 研究、高级生物统计学、机器学习、生物信息学和湿法实验室技术 发展，以提高口头和书面沟通和演示技能以及时间管理。

Project Summary/Abstract Over one in one hundred infants born are affected by neurodevelopmental disorders (NDDs), which can cause a host of debilitating symptoms including early-onset seizures, developmental delay, and intellectual disability. Application of whole exome/genome sequencing to examine NDD has revealed many disease-causing variations, but in a substantial proportion of cases the genetic cause remains undetermined. The analysis of deep-intronic non-coding variants has provided diagnosis when coding variation cannot be implicated in disease. For example, intronic variants in the “20N” poison exon of SCN1A results in loss of function, causing Dravet Syndrome, an NDD causing prolonged seizures in the first year of life. Poison exons are a mechanism of gene regulation whereby a premature termination codon caused by inclusion of the poison exon induces nonsense- mediated decay of the mRNA transcript. Preliminary analysis has shown that poison exon inclusion (PEI) in SCN1A and SCN8A decreases during embryonic brain development, inversely proportional to total RNA expression. The brain is a hotbed of alternative splicing during early neuronal development therefore it is plausible, if not probable that novel poison exons remain uncharacterized. The overall hypothesis of this proposal is that PEI is a mechanism to differentiallyregulate gene expression during earlyneuronal development, and intronic variants affecting PEI in genes of developmental importance contribute to disease phenotype. In Aim 1, the sodium and calcium voltage-gated channel alpha subunit families will be assessed to detect and characterize novel poison exons. Known and novel PEI will then be determined in the developing brain using publicly available RNA-seq experiments. In Aim 2 this analysis will be scaled up over 100-fold to detect and assess PEI in over 3000 genes associated with NDD and Autism Spectrum Disorder (ASD) in the developing brain and the driving forces of PEI evolution will be assessed. Comparative analysis of poison exons will be conducted using k-means hierarchical clustering and unsupervised random forest machine learning methodologies to determine prime biological factors driving PEI utilization. In Aim 3, intronic variants will be analyzed in probands of NDD and ASD in regions of known and hypothetical PEI and the affect these variants have on disease phenotype will be computationally assessed. These experiments will reveal new insights into alternative splicing in the developing brain and provide potential genetic explanation of currently unknown NDD disease phenotype. The research will be conducted at the HudsonAlpha Institute for Biotechnology, which allows developing scientists to hone skills in a dynamic, hands-on environment with supplemental training courses as necessary. Besides the research outlined in the proposal, training will include courses in conduct of responsible research, advanced biostatistics, machine learning, bioinformatics, and wet lab techniques next to personal development to improve oral and written communication and presentation skills as well as time management.