中国肌萎缩侧索硬化患者DIAPH3基因筛查及其遗传、细胞损伤机制研究

Amyotrophic lateral sclerosis (ALS) is a kind of fatal neurodegenerative disease. The cytoskeletal abnormalities are reported to correlated with pathogenesis of ALS. DIAPH3 gene plays an important role in the formation of cytoskeleton, including filaments and microtubules, and regulates the expression of multiple ALS causative genes. The applicant identified several ALS patients, including familial and sporadic cases, carrying possibly damaging mutations in DIAPH3. However, direct correlations and related mechanism between DIAPH3 and ALS are blurred. Therefore the presented study intends to explore the role of DIAPH3 gene in the pathogenesis of ALS: by using the next generation sequencing technology, this study is about to identify the prevalence and clinical features of DIAPH3 mutation carriers; by using the genetic burden test, the study will explore genetic statistical correlation between the DIAPH3 and ALS, especially for the genetic variants in the specific pathways, such as genes related to formation or functional regulation of the cytoskeleton; by observing motor neuron cell lines with DIAPH3 mutations expressed, the study is going to verify the possible cellular mechanism of DIAPH3 in ALS pathogenesis. This study will provide important insights for the future genetic diagnosis and treatment of ALS. The research team has rich experience in genetic study and related molecular mechanisms especially for ALS. It is expected that this study could achieved advanced and outstanding results even in the worldwide.

肌萎缩侧索硬化(ALS)是一种致死性的神经变性病。细胞骨架异常与ALS发病密切关联。DIAPH3对细胞骨架中微丝及微管形成有重要影响，调控多个ALS致病基因表达。申请人已发现数个携带DIAPH3致病突变的中国ALS家系及散发病例，高度提示该基因与ALS相关。但DIAPH3与ALS直接关联不明确且发病机制研究空缺。故本研究拟系统探讨DIAPH3基因与ALS发病的直接关系和机制：用二代测序方法，明确DIAPH3在中国ALS发病人群中的基因频率、突变携带患者的临床特点；对DIAPH3和与细胞骨架功能、ALS相关的多基因变量进行变异负担分析，明确DIAPH3突变与ALS发病的遗传相关性；观察DIAPH3突变在运动神经元细胞株VSC4.1表达对细胞活性及对细胞骨架、泛素化蛋白影响，印证发病机制。本研究将对ALS的基因诊断和治疗提供重要指导意义。申请人研究经验丰富、条件成熟，预期成果能具备先进水平。

肌萎缩侧索硬化(ALS)是一种致死性的神经变性病。细胞骨架异常与ALS发病密切关联。DIAPH3对细胞骨架中微丝及微管形成有重要影响，调控多个ALS致病基因表达。但DIAPH3与ALS直接关联不明确且发病机制研究空缺。. 故本研究利用遗传学及分子生物学方法系统探讨DIAPH3基因与ALS发病的直接关系和机制:用二代测序方法，明确了DIAPH3在中国ALS发病人群中的基因频率、突变携带患者的临床特点。并对DIAPH3和与细胞骨架功能、ALS相关的多基因变量进行变异负担分析，明确DIAPH3突变与ALS发病的遗传相关性。在VSC4.1细胞系表达hDIAPH3WT及hDIAPH3突变型，初步探索DIAPH3在ALS发病中的可能机制和运动神经元细胞改变，观察DIAPH3突变在运动神经元细胞株VSC4.1表达对细胞活性及对细胞骨架、泛素化蛋白影响，印证发病机制。. 本课题目前已建立和补充、完善ALS的数据库和遗传资料库，3年内新增纳入约1000例ALS患者数据，建立大样本队列（完善患者近600例全外显子测序数据）,掌握ALS常见致病类型的流行病学资料,追踪自然病史,分析中国汉族人群与欧美国家流行病学的异同,为发病机制、量表及治疗的研究搭建良好的平台（包括研究性网络平台，www.chalsr.net）。完善了部分样本全基因组外显子测序及筛查，对部分患者进行神经、肌肉病理的检测。. 课题奠定了一定基础，确立及改良了部分高通量分析方法、通路分析等关键技术，明确了进一步研究的可行性。本课题发表系列SCI期刊文章6篇，核心期刊2篇。课题主持人以第一作者、责任作者身份分别发表工作于《Nature Communications》，《Journal of Neurology, Neurosurgery, and Psychiatry》，《Neurobiology Aging》等较高水平期刊，同时课题协同发布一项开源软件（https://github.com/gc5k/GEAR），成果水平良好。

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