Development of an information-rich assay for C9ORF72 as a test for ALS and FTD

项目摘要 该项目的长期目标是开发、验证和商业化一种用于改进分析的分析方法 C9orf72，9号染色体上的一个基因，与额颞痴呆(FTD)和肌萎缩侧索硬化症相关 硬化症(ALS)。富含鸟嘌呤和胞嘧啶的六核苷酸重复序列(GGGGCC)在非编码区的扩增 C9orf72基因与39%的家族性ALS相关。这种扩张也出现在25%的家族性 额颞叶痴呆(FTD)病例，以及7%的散发性ALS和6%的散发性FTD。C9orf72 区域的大小很难准确确定，因为在受影响的个体中，大多数扩增的长度都是&gt；700重复。 目前的检测通常依赖于“自制的”基于聚合酶链式反应的检测方法来准确测定35个重复序列，或者， 另外，对粗制的&gt；35重复进行了Southern杂交分析。我们已经开发了一种解决方案，以实现高 对于长达145次的短(35个重复)重复区域，吞吐量、可靠、灵敏和准确的大小 反射试验中的重复和长扩增(146到至少~800次)。建议的化验提供了一种 基于重复启动检测平台(AmplideX®FMR1 PCR)解决这些技术挑战 Asurgen已经开发并成功商业化了脆性X综合征，这是一种CGG三联体重复障碍。 我们将继续利用&gt；6年在优化GC的高性能诊断分析方面的经验。 丰富的重复序列，为C9orf72开发一种准确和可靠的诊断测试。这一第二阶段的资金 将支持完成测试开发所需的努力。这项建议的具体目标是： 目的1.整合一种新型的工程聚合酶系统，以实现极高的处理速度和可靠性 扩增&gt；800个六核苷酸重复序列。 目标2.在支持C9orf72的优化工作流程中开发和集成一组控制和标准 测试。 目标3.在用户友好的应用程序中开发数据分析管道和报告工具，以便 快速准确识别C9orf72重复数。 目标4.评估将工作流程和控制(目标1和2)与 分析软件(AIM 3)。用细胞系和回溯性临床标本验证集成系统。 开发一种改进的、信息丰富的C9orf72检测方法将是有用的筛查和 ALS和FTD的诊断测试，以及识别中间和/或扩展的临床研究工具 可能与其他形式的老年性神经变性相关的重复大小。此外，这一检测方法 可以促进对已知和新的基因-表型关联的理解，并使机会 用于靶向治疗和临床试验。

Project Summary The long term goal of this project is to develop, validate, and commercialize an assay for improved analysis of C9orf72, a gene on chromosome 9 that is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Expansion of a guanine and cytosine rich hexanucleotide repeat (GGGGCC) in the non-coding region of C9orf72 is associated with 39% of familial ALS. The expansion also appears in 25% of familial frontotemporal dementia (FTD) cases, as well as 7% of sporadic ALS and 6% of sporadic FTD. The C9orf72 region is difficult to size accurately because most expansions in affected individuals are >700 repeats in length. Currently testing typically relies on “homebrew” PCR-based assays for accurate sizing of <35 repeats, or, separately, Southern blot analysis for crude sizing of >35 repeats. We have developed a solution to enable high throughput, reliable, sensitive, and accurate sizing of the repeat region for both short (<35 repeats) up to 145 repeats, and long expansions (146 to at least ~800 repeats) in a reflex assay. The proposed assay offers a solution to these technical challenges based on the repeat-primed assay platform (AmplideX® FMR1 PCR) that Asuragen has developed and successfully commercialized for fragile X syndrome, a CGG triplet repeat disorder. We will continue to leverage >6 years of experience in optimizing high performance diagnostic assays for GC- rich repeat sequences to develop an accurate and robust diagnostic test for C9orf72. Funding for this Phase II will support the efforts necessary to complete the development of the test. The specific aims of this proposal are: Aim 1. Integrate a novel engineered PCR polymerase system to achieve extreme processivity and reliable amplification of >800 hexanucleotide repeats. Aim 2. Develop and integrate a set of controls and standards in an optimized workflow that supports C9orf72 testing. Aim 3. Develop a data analysis pipeline and reporting tools within a user-friendly application that allows rapid and accurate identification C9orf72 repeat number.   Aim 4. Evaluate an assay system that integrates the workflow and controls (Aims 1 and 2) with the analysis software (Aim 3). Validate the integrated system with cell lines and retrospective clinical samples. The development of an improved, information-rich assay for C9orf72 will be useful as a screening and diagnostic test for ALS and FTD as well as a clinical research tool to identify intermediate and/or expanded repeat sizes that are potentially relevant to other forms of age-onset neurodegeneration. In addition, this assay can further the understanding of known and novel genotype-phenotype associations and enable opportunities for targeted therapeutics and clinical trials.