Characterisation of biological pathways dysregulated in amyotrophic lateral sclerosis (ALS) cases with a hexanucleotide repeat expansion of C9ORF72.

肌萎缩性侧索硬化症（ALS）是一种迅速进展的悲剧性神经退行性疾病。最近发现，基因C9ORF72的扩增导致了相当大比例的ALS。C9ORF72在疾病和健康中的功能目前尚不清楚。该项目的目的是确定与C9ORF72扩增相关的疾病机制。这将是更广泛地开发ALS治疗药物的重大进展。为了发挥功能，基因（如DNA）必须以RNA的形式表达。据推测，C9ORF72扩增通过破坏细胞核中RNA的组装而引起疾病。这是由申请人进行的试点工作支持的。研究RNA组装疾病的理想方法是基因表达谱（GEP）；这是一种测量样品中不同种类RNA水平的技术。GEP将在我们当地组织库中的C9ORF72患者和对照组的死后组织中进行；以及来自MNDA国家DNA银行和相关淋巴母细胞样细胞系（LCL）资源的C9ORF72患者和对照的血液细胞。此外，专门用于识别错误组装RNA的技术将应用于来自C9ORF72患者的皮肤细胞和我们当地生物库中的对照；这将在利物浦MRC高通量测序（HTS）中心进行（见支持信）。通过与Al-Chalabi教授的合作，将获得MNDA国家DNA库和相关LCL资源中C9ORF72扩增样本的身份（见支持信）。该资源包含大量样本，因此适用于GEP研究，不仅适用于C9ORF72扩增的患者，也适用于疾病快速进展或缓慢进展的患者。这可能有助于发现作为潜在治疗靶点的疾病调节剂。疾病调节剂的存在得到了申请人试点工作的支持，表明与C9ORF72扩增相关的疾病是显著可变的。通过与Al-Chalabi教授的合作，已经获得了同一组患者DNA变化的数据，并将与GEP数据相关联。因此，疾病调节剂将在DNA和RNA水平上被理解。已经提出的与C9ORF72相关的疾病修饰因子是扩展的大小，已被证明在患者之间是可变的。这是其他类似疾病的重要疾病调节剂。扩增将使用来自MNDA国家DNA银行的DNA样本，使用一种称为Southern blotting的技术进行测定。扩大的大小与疾病的各个方面，如生存时间之间的关系将被检查。细胞模型是研究疾病机制的有用工具。除了用于来自C9ORF72扩增患者的GEP皮肤细胞外，还将进行独立研究，以确定它们与对照皮肤细胞的差异。这项技术已成功应用于ALS和其他神经系统疾病的研究。这将部分受到上述其他分析结果的指导，以提供对这些发现的验证。关于C9ORF72 ALS的另一个观察结果是，C9ORF72基因编码的蛋白质在扩增患者中以较低的浓度存在。这可能是与RNA组装影响共存的另一种疾病机制。这将通过在实验室培养的神经细胞模型中观察阻止C9ORF72蛋白表达的效果来研究。这项工作将主要由申请人进行，但会得到SITraN（谢菲尔德转化神经科学研究所）功能基因组学、计算生物学和细胞生物学团队的协助。

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and tragic neurodegenerative condition. It has been recently been discovered that an expansion of the gene C9ORF72 causes a relatively large proportion of ALS. The function of C9ORF72 in disease and health is currently unknown. The aim of this project is to determine disease mechanisms associated with the C9ORF72 expansion. This will be a major advance towards the development of therapeutic agents for ALS more widely. In order to become functional, genes (as DNA) must be expressed in a RNA form. It has been postulated that the C9ORF72 expansion causes disease by disruption of the assembly of RNA in the cell nucleus. This is supported by pilot work carried out by the applicant. The ideal method for studying a disease of RNA assembly is gene expression profiling (GEP); this is a technique which measures the levels of various species of RNA in a sample. GEP will be carried out on post-mortem tissue from C9ORF72 patients and controls within our locally held tissue bank; and on cells from the blood of C9ORF72 patients and controls obtained from the MNDA National DNA bank and associated lymphoblastoid cell line (LCL) resource. Furthermore technology specialised for identifying misassembled RNA will be applied to skin cells from C9ORF72 patients and controls within our locally held biobank; this will be carried out in the MRC High-Throughput Sequencing (HTS) hub in Liverpool (see letter of support). The identity of samples with the C9ORF72 expansion within the MNDA National DNA bank and associated LCL resource will be obtained through collaboration with Professor Al-Chalabi (see letter of support). This resource contains a large number of samples and therefore is suitable for a GEP study, not only of patients with the C9ORF72 expansion but of such patients with either rapidly progressive or slowly progressive disease. This may facilitate the discovery of modifiers of disease which are potential therapeutic targets. The presence of disease modifiers is supported pilot work by the applicant showing that disease associated with the C9ORF72 expansion is significantly variable. Data about DNA changes in the same group of patients has already been obtained through collaboration with Professor Al-Chalabi and will be correlated with the GEP data. Thus disease modifiers will be understood at the DNA and the RNA level. A modifier of disease associated with C9ORF72 which has aready been proposed is the size of the expansion which has been shown to be variable between patients. This is a significant disease modifier in other similar diseases. The expansion will be sized using DNA samples from the MNDA National DNA bank using a technique called Southern blotting. The relationship between the size of the expansion and various aspects of disease such as survival time will be examined.Cell models are a useful tool for studying disease mechanisms. As well as being used in GEP skin cells from patients with the C9ORF72 expansion will be studied independently to determine how they differ to skin cells from controls. This technique has been used successfully in the study of ALS and other neurological diseases. This will be guided in part by the results of the other analyses described above to provide a validation of those discoveries. Another observation about C9ORF72 ALS is that the protein encoded by the C9ORF72 gene is present at a reduced concentration in patients with the expansion. This may be an additional disease mechanism co-existing with an effect on RNA assembly. This will be investigated by observing the effect of preventing expression of the C9ORF72 protein in a model consisting of nerve cells grown in the lab.This work will be carried out primarily by the applicant but with assistance from the Functional Genomics, Computational Biology and Cell Biology teams within SITraN (Sheffield Institute for Translational Neuroscience).