Genome-Wide Somatic Mutation in Alzheimer's Disease Pathogenesis Using Single Neuron Analysis

项目总结/摘要 这份NIH K08提案描述了一项为期五年的神经退行性疾病职业发展培训计划， 疾病基因组学研究。Michael米勒博士已完成解剖病理学的临床住院医师培训， 布里格姆妇女医院（BWH）和波士顿儿童医院（BCH）神经病理学奖学金 在哈佛医学院（HMS），并将着手这项研究计划，培养一个独立的 学术研究生涯，探讨神经退行性疾病的发病机制。 在这个培训计划中，米勒博士将发展单细胞基因组学，下一代 测序数据分析、非肿瘤性人类体细胞突变的生物学解释及其应用 对神经退行性疾病机理研究的基因组学方法。他的导师克里斯托弗博士 沃尔什（HMS的神经学教授和BCH的HHMI研究员）是人类神经学的领导者 疾病遗传学和基因组学。他的实验室在人类遗传学、神经学 疾病基因定位、单细胞基因组学和大型数据集分析。沃尔什医生建立了一个长期的 指导其他学员在生物医学研究方面取得成功的记录。另外，米勒博士 已经召集了一组具有互补专业知识的合作者，以及一个咨询委员会， 在指导医生科学家开发独立研究项目方面拥有丰富的经验。他将 以教学课程和在国际和国家会议上介绍工作来补充这种培训。 拟议研究计划的主要科学目标是研究神经元体细胞的作用， 阿尔茨海默病（AD）中的突变。米勒博士提供的初步数据表明，AD中的神经元显示， 以升高的体细胞单核苷酸变体（sSNV）形式的大量基因组损伤， 控制神经元。该提案将通过研究这一发现的分布情况， AD脑及其与AD细胞病理生理学中已知关键事件的关系：氧化应激和Tau 错误折叠这一提议的中心假设是异常的Tau和氧化损伤驱动体细胞凋亡。 在AD中的突变，构成了疾病发病机制的细胞损伤级联。 单细胞全基因组测序（scWGS）将用于解决三个独立但相关的 关于体细胞突变在AD中的作用的问题：（1）将在脑的3个区域中比较sSNV负荷 在AD的不同阶段受到影响，以评估sSNV与疾病病理进展的关系;（2） 将确定AD的体细胞突变特征，以确定sSNV的近因，沿着 直接测试氧化DNA中的推定候选物;以及（3）具有更大Tau积累的神经元将被 检查sSNV以评估细胞病理负荷与体细胞突变之间的关系。 这些研究将检查AD病因学中的一种新的致病事件，这对以下领域具有重要意义： 神经退行性疾病发病机制和基因组学。

PROJECT SUMMARY / ABSTRACT This NIH K08 proposal describes a five-year career development training program in neurodegenerative disease genomics research. Dr. Michael Miller has completed clinical residency in Anatomic Pathology and fellowship in Neuropathology at Brigham and Women’s Hospital (BWH) and Boston Children’s Hospital (BCH) at Harvard Medical School (HMS), and will embark on this research program to train for an independent academic research career to investigate the pathogenesis of neurodegenerative disease. In this training program, Dr. Miller will develop expertise in single cell genomics, next-generation sequencing data analysis, biologic interpretation of non-neoplastic human somatic mutations, and application of genomic approaches toward neurodegenerative disease mechanistic inquiry. His mentor, Dr. Christopher Walsh (a Professor of Neurology at HMS and HHMI Investigator at BCH), is a leader in human neurologic disease genetics and genomics. His laboratory has extensive experience in human genetics, neurologic disease gene mapping, single cell genomics, and analysis of large data sets. Dr. Walsh has established a long track record for mentoring other trainees to successful careers in biomedical research. In addition, Dr. Miller has assembled a group of collaborators with complementary expertise, and an Advisory Committee with extensive experience in mentoring physician-scientists to develop independent research programs. He will supplement this training with didactic courses and presentation of work at international and national meetings. The primary scientific objective of the proposed research plan is to study the role of neuronal somatic mutation in Alzheimer’s disease (AD). Dr. Miller provides pilot data indicating that neurons in AD show substantial genome damage in the form of elevated somatic single nucleotide variants (sSNV) compared to control neurons. The proposal will examine the disease context for this finding, by studying its distribution in the AD brain and relationship to known key events in AD cellular pathophysiology: oxidative stress and Tau misfolding. The central hypothesis of this proposal is that aberrant Tau and oxidative damage drive somatic mutation in AD, constituting a cascade of cellular damage that underlies disease pathogenesis. Single cell whole genome sequencing (scWGS) will be employed to address three independent but related questions about the role of somatic mutation in AD: (1) sSNV burden will be compared in 3 areas of the brain affected at different stages in AD, to assess sSNV relationship to disease pathologic advancement; (2) Somatic mutational signatures will be determined for AD, to identify the proximate causes of sSNV, along with direct testing for a putative candidate in oxidized DNA; and (3) neurons with greater Tau accumulation will be examined for sSNV to assess the relationship between cellular pathologic burden and somatic mutations. These studies will examine a novel pathogenic event in AD etiology, significant for the fields of neurodegenerative disease pathogenesis and genomics.