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

C9ORF72 六核苷酸重复扩增导致肌萎缩侧索硬化症 (ALS) 病例中生物途径失调的表征。

• 批准号: MR/K003771/1
• 负责人: Johnathan Cooper-Knock
• 金额: $ 24.48万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Fellowship
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FK003771%2F1
• 关键词: Characterisation; biological; pathways; dysregulated; amyotrophic

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).

肌萎缩侧索硬化症(ALS)是一种进展迅速的悲剧性神经退行性疾病。最近发现，C9ORF72基因的扩展导致了相对较大比例的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扩展相关的疾病机制。这将是朝着更广泛的ALS治疗剂发展的重大进展。为了发挥功能，基因(如DNA)必须以RNA的形式表达。有人推测，C9ORF72的扩张是通过破坏核内RNA的组装而导致疾病的。这一点得到了申请人开展的试点工作的支持。研究RNA组装疾病的理想方法是基因表达谱(GEP)；这是一种测量样本中各种RNA水平的技术。GEP将在我们本地保存的组织库中对C9ORF72患者和对照组的身体组织进行，对来自MNDA国家DNA库和相关的淋巴母细胞系(LCL)资源的C9ORF72患者和对照组的血液细胞进行GEP。此外，专门用于识别错误组装的RNA的技术将应用于C9ORF72患者和我们本地持有的生物库中的对照皮肤细胞；这将在利物浦的MRC高通量测序(HTS)中心进行(见支持函)。通过与Al-Chalabi教授合作，将获得MNDA国家DNA银行内具有C9ORF72扩展的样本和相关的LCL资源(见支持函)。该资源包含大量样本，因此适用于GEP研究，不仅适用于C9ORF72扩张型患者，也适用于此类疾病进展迅速或缓慢的患者。这可能有助于发现潜在的治疗靶点的疾病修饰物。疾病修饰物的存在得到了申请人的支持，试点工作表明，与C9ORF72扩展相关的疾病具有显著的变异性。关于同一组患者DNA变化的数据已经通过与Al-Chalabi教授的合作获得，并将与GEP数据相关联。因此，疾病修饰物将在DNA和RNA水平上得到理解。已经提出的与C9ORF72相关的疾病的修饰物是扩张的大小，已被证明在患者之间是可变的。在其他类似的疾病中，这是一个重要的疾病修饰物。扩增的大小将使用来自MNDA国家DNA库的DNA样本，使用一种名为Southern blotting的技术。我们将研究扩张的大小与疾病的各个方面之间的关系，如存活时间。细胞模型是研究疾病机制的有用工具。除了用于GEP，C9ORF72扩增患者的皮肤细胞还将进行独立研究，以确定它们与对照组皮肤细胞的不同之处。这项技术已经成功地应用于ALS和其他神经疾病的研究。这将在一定程度上受到上述其他分析结果的指导，以验证这些发现。关于C9ORF72 ALS的另一个观察结果是，C9ORF72基因编码的蛋白质在扩张的患者中浓度降低。这可能是一种额外的疾病机制，与对RNA组装的影响共存。这将通过观察在实验室培养的神经细胞组成的模型中防止C9ORF72蛋白表达的效果来进行研究。这项工作将主要由申请人进行，但需要SITraN(谢菲尔德翻译神经科学研究所)的功能基因组学、计算生物学和细胞生物学团队的协助。

项目成果

Comparison of blood RNA extraction methods used for gene expression profiling in amyotrophic lateral sclerosis.

• DOI: 10.1371/journal.pone.0087508
• 发表时间: 2014
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Bayatti N;Cooper-Knock J;Bury JJ;Wyles M;Heath PR;Kirby J;Shaw PJ
• 通讯作者: Shaw PJ

The Spectrum of C9orf72-mediated Neurodegeneration and Amyotrophic Lateral Sclerosis.

• DOI: 10.1007/s13311-015-0342-1
• 发表时间: 2015-04
• 期刊: NEUROTHERAPEUTICS
• 影响因子: 5.7
• 作者: Cooper-Knock, Johnathan;Kirby, Janine;Highley, Robin;Shaw, Pamela J.
• 通讯作者: Shaw, Pamela J.

The widening spectrum of C9ORF72-related disease; genotype/phenotype correlations and potential modifiers of clinical phenotype.

• DOI: 10.1007/s00401-014-1251-9
• 发表时间: 2014-03
• 期刊: Acta neuropathologica
• 影响因子: 12.7
• 作者: Cooper-Knock J;Shaw PJ;Kirby J
• 通讯作者: Kirby J

Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy.

• DOI: 10.1007/s00401-015-1429-9
• 发表时间: 2015-07
• 期刊: Acta neuropathologica
• 影响因子: 12.7
• 作者: Cooper-Knock J;Higginbottom A;Stopford MJ;Highley JR;Ince PG;Wharton SB;Pickering-Brown S;Kirby J;Hautbergue GM;Shaw PJ
• 通讯作者: Shaw PJ

Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions.

• DOI: 10.1093/brain/awu120
• 发表时间: 2014-07
• 期刊: Brain : a journal of neurology
• 作者: Cooper-Knock J;Walsh MJ;Higginbottom A;Robin Highley J;Dickman MJ;Edbauer D;Ince PG;Wharton SB;Wilson SA;Kirby J;Hautbergue GM;Shaw PJ
• 通讯作者: Shaw PJ