High-Throughput Genomic Approaches to Identify ALS Genes

描述（由申请人提供）：肌萎缩侧索硬化症（ALS）是一种致命的运动神经元退行性疾病。虽然有10%的病例是常染色体显性遗传，但它主要是散发性的。胞质铜锌超氧化物歧化酶基因（SOD 1）突变占显性遗传病例的25%。对家族性和散发性ALS发病机制的深入研究来自于对SOD 1基因突变的研究。一些显性遗传的ALS基因已经被定位，但尚未确定。本项目的目标是应用高通量基因组学方法系统地鉴定新的ALS基因，并利用这些基因建立新的ALS转基因小鼠模型。这些努力将针对我们最近在9号、16号和20号染色体上发现的显性ALS基因座。本项目的具体目标是：（1）在小鼠-人体细胞杂交后代的细菌人工染色体（BAC）文库中克隆9-、16-和20-染色体连锁ALS的患病个体的疾病位点，（2）通过外显子测序筛选这三个ALS位点的候选基因;（3）使用高通量测序来确定跨越ALS基因座的BAC重叠群的基因组序列;（4）确认候选突变的疾病关联;（5）通过显微注射在目的1-4中鉴定的基因中含有突变的BAC来工程化ALS的小鼠敲入模型。重要性：这些研究将是重要的，因为：（A）该项目可能作为系统识别其他孟德尔疾病特征的新范例。(B)这些研究将阐明运动神经元存活的关键途径。相同的途径可能参与散发性ALS。(C)了解这种疾病的分子病理生理学对于合理设计治疗干预措施至关重要。(D)在这些实验中鉴定的基因可用于创建ALS的其他动物和细胞模型，从而改进药物设计和筛选。(E)ALS细胞死亡途径的特征可能会为阿尔茨海默病和帕金森病等其他神经退行性疾病的机制提供见解。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a fatal degenerative disorder of motor neurons. It is predominantly sporadic in occurrence, although 10% of cases are inherited as autosomal dominant traits. Mutations in the cytosolic copper-zinc superoxide dismutase gene (SOD1) account for 25% of dominantly-inherited cases. Considerable insight into the pathogenesis of both familial and sporadic ALS has been derived from studies of SOD 1 gene mutations. Several more dominantly-inherited ALS genes have been mapped but not yet identified. The goal of this project is to apply high-throughput genomic methodology systematically to identify novel ALS genes, and to develop new transgenic mouse models of ALS with these genes. These efforts will target the dominant ALS loci that we have recently identified on chromosomes 9, 16, and 20. The Specific Aims of this project are to: (1) Clone the disease loci from affected individuals with chromosome 9-, 16-, and 20- linked ALS in bacterial artificial chromosome (BAC) libraries prepared from mouse-human somatic cell hybrids haploid for the chromosomes of interest; (2) Screen candidate genes from the three ALS loci by exon sequencing; (3) Use high-through-put sequencing to determine the genomic sequence of the BAC contig spanning the ALS loci; (4) Confirm the disease association of candidate mutations; (5) Engineer mouse knock-in models of ALS by microinjection of BACs harboring mutations in the genes identified in Aims 1-4. Significance: These studies will be important because: (A) This project may serve as a new paradigm for the systematic identification of other Mendelian disease traits. (B) These studies will elucidate pathways critical for the survival of motor neurons. The same pathways may be involved in sporadic ALS. (C) An understanding of the molecular pathophysiology of this disease is essential to the rational design of therapeutic interventions. (D) The genes identified in these experiments can be used to create additional animal and cell models of ALS, allowing improved drug design and screening. (E) The characterization of cell death pathways in ALS may provide insights into mechanisms involved in other neurodegenerative disorders such as Alzheimer's and Parkinson's diseases.