阿尔茨海默病PLD3基因深度测序及其罕见突变的致病机制研究

Recently, a whole-exome sequencing (WES) study has shown that a rare variant of the phospholipase D3 (PLD3) gene confers a doubled risk for late-onset Alzheimer’s disease (LOAD) in Caucasian population. Due to the differences in genetic background, whether PLD3 is still associated with LOAD risk in Han Chinese remains largely unclear; and few studies focus on the detailed mechanisms of the rare mutations of PLD3 in AD pathogenesis. In our preliminary studies, we have provided the first evidence that a novel missense mutation (R356H) and a splicing mutation (c.1020-8G>A) in exon 11 of PLD3 were associated with LOAD risk in Han Chinese. Our current researches intend to further map PLD3 to fully identify risk variants for LOAD in a large Han Chinese cohort. In addition, the Minigene plasmids for c.1020-8G>A mutation was constructed and transfected into cells, and the effect of c.1020-8G>A mutation on pre-mRNA splicing was analyzed. R356H mutation was introduced into pcDNA3.1/PLD3 vector, and expression characteristics of PLD3 in transfected cells were measured. Meanwhile, the regulatory roles of PLD3 mutations in Aβ, neuronal and synaptic pathology were confirmed in lentivirus stably transfected cell lines. This project will provide theoretic support for early genetic diagnosis of AD in Han Chinese, and offer more information for molecular therapy via uncovering its underlying pathogenesis.

高加索人群二代测序发现PLD3为LOAD新的致病基因，其罕见突变增加患病风险2倍。由于各种族人群遗传背景存在差异，目前PLD3是否与汉族人群LOAD风险相关，及其突变导致AD的分子机制尚未明确。我们前期已建立汉族人群LOAD资源库，并率先在PLD3第11号外显子检测到全新错义突变R356H和剪接位点突变c.1020-8G>A。本项目在此基础上，拟对PLD3除11号外显子外其他关键区域进行二代高通量测序，精确定位所有变异位点频率分布，筛选出与汉族人群LOAD风险相关的变异位点。此外，拟构建携带c.1020-8G>A突变Minigene质粒，及携带R356H突变真核表达载体，瞬时转染细胞后，研究上述突变对其mRNA前体剪接以及编码蛋白转录表达的影响。进一步构建慢病毒介导的稳转细胞模型，研究PLD3突变对Aβ病理、神经元及突触的影响，阐明PLD3突变导致AD的分子机制，并为AD诊疗提供全新靶点。

本项目前期建立了汉族人群大样本LOAD资源库，完成了PLD3第11号外显子测序工作，并率先在汉族人群LOAD患者中发现了全新的错义突变R356H以及剪接位点变异c.1020-8G>A。在此基础上，应用二代高通量测序技术对汉族人群PLD3除11号外显子以外的其他关键区域进行深度测序，首次发现PLD3基因I163M突变也可显著增加AD发病风险，从而全面揭示了汉族人群PLD3与LOAD易感性的关系。我们进一步对PLD3基因新发现突变在AD发病中的作用机制进行了深入研究：通过构建携带I163M及R356H突变真核表达载体，转染细胞后显示，上述突变尚未影响PLD3 mRNA以及蛋白转录表达水平，但使PLD3蛋白活性显著减低。进一步通过构建慢病毒介导的稳转细胞模型，首次证实PLD3新发现突变显著影响PLD3活性，低活性PLD3通过下游mTOR-自噬通路，影响Aβ的清除过程进而参与AD。本项目的研究成果阐明了PLD3突变导致AD的分子机制，并为AD诊疗提供了全新靶点。本项目共撰写SCI收录论文3篇，其中2篇已在JAD杂志发表，另外1篇已投稿至Front. Neurosci.杂志，审稿人均已同意发表。在本项目的支持下，项目负责人于2016 年入选山东省“泰山学者”青年专家。本项目研究成果在国内外学术会议上发言并获得国际学术奖励，培养博士1名，硕士4名。并荣获中华医学会青年科技奖1项，山东省科技进步二等奖1项，青岛市科技进步二等奖1项。本项目研究成果已在多家大型三甲医院中推广应用，为后续深入探讨AD新发现突变的致病机制和防治措施奠定了坚实基础，并取得了显著的社会效益。