Defining the gene regulatory roles of non-coding variants in the pathogenesis of autism

摘要 在这个博士前奖学金的建议，我将被训练为一个未来的医生，科学家与我自己的 在基因组学，计算生物学和神经科学的接口独立的研究计划。 在我的MD/PhD培训期间，我将由两位医生科学家John Greally博士（医学博士）共同指导。 基因组学）和巴勃罗卡斯蒂略（神经学），解决一个问题，是及时与即将广泛的 全基因组测序（WGS）在临床诊断中的应用-我们如何解释非- 当病人出现医疗问题时，编码大部分人类基因组？ 我将集中讨论自闭症，因为自闭症是一种代表了相当大比例的患者接受医疗检查的疾病。 遗传学服务，其中有来自数千个家庭的广泛的WGS信息。尽管这些财富 在研究测序中，只有一小部分自闭症患者获得了积极的结果， 诊断外显子组或WGS。我认为，目前有限的诊断成功率主要是由于我们的 无法解释这些患者的非编码大部分基因组中的致病变异。通过 提高我们对非编码变异的洞察力，我们将能够为更多的人提供诊断信息。 家庭寻求比现在更好的答案。 我的策略是关注未受影响的父母所生的自闭症后代的新生变异（DNV）。我 假设是当DNV出现在细胞类型的顺式调节区时，它们可能是致病的 调解自闭症。因此，该项目基于计算基因组学基础，使用WGS和 DNV电话来自数千个家庭的大型数据集，这些家庭中有一名成员患有自闭症。在我的初步调查中 数据，我表明，在自闭症个体的DNV富集在神经胶质细胞和神经元细胞的顺式调节位点， 特别是，在基因已知是自闭症的病因。在我的项目中，我将更多地测试这些关联 并将为功能测试定义一组高置信度的DNV。 将进行两种类型的功能测试。一个将测试DNV是否改变分子基因组 特性，包括染色质可及性和基因表达。第二个将测试生理 细胞的特性。为了产生合适的细胞进行测试，我计划使用诱导多能干细胞 在一些实施方案中，诱导多能干细胞（iPSC）在体外分化为GABA能神经元和星形胶质细胞。通过进行CRISPR介导的 通过在iPSC中进行基因组编辑，我可以产生具有候选致病性DNV的细胞，并测试它们是否 影响细胞特性，如树突发生、突触发生和电生理学， 相信这些DNV具有致病作用。 我将有幸接受复杂计算、干细胞和神经科学方面的培训 技术，在各自领域的两位领导人的指导下，作为全面培训计划的一部分， 装备我成为独立的物理学家，科学家，我渴望在我的职业生涯。

ABSTRACT In this Predoctoral Fellowship proposal, I will be trained for a future as a physician-scientist with my own independent research program at the interface of genomics, computational biology, and neuroscience. During my MD/PhD training, I will be co-mentored by two physician-scientists, Drs. John Greally (Medical Genomics) and Pablo Castillo (Neurology), addressing a question that is timely with the imminent widespread use of whole genome sequencing (WGS) in clinical diagnostics – how do we interpret variants in the non- coding majority of the human genome when a patient presents with a medical problem? I will focus on autism, as a condition that represents a substantial proportion of patients seen by medical genetics services, for which there is extensive WGS information from thousands of families. Despite this wealth of research sequencing, only a small minority of individuals with autism receive a positive outcome of diagnostic exome or WGS. I propose that the currently limited diagnostic success rates are mostly due to our inability to interpret pathogenic variants in the non-coding majority of the genome of these patients. By improving our insights into non-coding variants, we will be able to offer diagnostic information to many more families seeking answers than currently. My strategy is to focus on de novo variants (DNVs) in offspring with autism born to unaffected parents. My hypothesis is that DNVs can be pathogenic when they occur in the cis-regulatory regions of cell types mediating autism. The project is therefore based on a computational genomics foundation, using WGS and DNV calls from large datasets from thousands of families who have a member with autism. In my preliminary data, I show that DNVs in individuals with autism are enriched at cis-regulatory loci in glial and neuronal cells in particular, and at genes known to be causative for autism. In my project, I will test these associations more rigorously, and will define a high-confidence set of DNVs for functional testing. Two types of functional testing will be performed. One will test whether the DNVs alter molecular genomic properties, including chromatin accessibility and gene expression. The second will test the physiological properties of the cells. To generate the appropriate cells for testing, I plan to use induced pluripotent stem cells (iPSCs) that are in vitro differentiated to GABAergic neurons and astrocytes. By performing CRISPR-mediated genomic editing in the iPSCs, I can generate cells with the candidate pathogenic DNVs, and test whether they have effects on cellular properties like dendritogenesis, synaptogenesis, and electrophysiology, increasing the confidence that these DNVs have pathogenic effects. I will have the privilege of getting training in sophisticated computational, stem cell and neuroscience techniques, under the guidance of two leaders in their fields, as part of a comprehensive training plan that will equip me to become the independent physician-scientist I aspire to be in my career.