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

开发信息丰富的 C9ORF72 检测方法作为 ALS 和 FTD 检测

• 批准号: 8782204
• 负责人: GARY J LATHAM
• 金额: $ 22.25万
• 依托单位: ASURAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8782204
• 关键词: Affect; Age of Onset; Agreement; Amyotrophic Lateral Sclerosis; Biological Assay; Blinded; C9ORF72; CGG repeat; Cell Line; Cells; Chromosomes, Human, Pair 9; Clinical; Clinical Research; Cytosine; DNA; Detection; Development; Diagnosis; Diagnostic; Diagnostic tests; Disease; Economic Burden; FMR1; Familial Amyotrophic Lateral Sclerosis; Foundations; Fragile X Syndrome; Frontotemporal Dementia; Functional RNA; Funding; Gene Mutation; Genes; Genetic; Genomic DNA; Genotype; Goals; Guanine; Individual; Laboratories; Length; Link; Methods; Methylation; Molecular Diagnostic Testing; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Performance; Phase; Phenotype; Process; Publishing; Sampling; Solutions; Southern Blotting; Testing; Therapeutic; Therapeutic Clinical Trial; Trinucleotide Repeats; Tube; Work; base; commercialization; experience; improved; novel; public health relevance; repository; screening; tool; validation studies

DESCRIPTION (provided by applicant): The long term goal of this project is to develop, validate, and commercialize an assay to improve screening of C9ORF72, a gene on chromosome 9 that is linked to frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Expansions of a guanine and cytosine rich hexanucleotide repeat (GGGGCC) in the non-coding region of C9ORF72 is associated with up to 67 percent of familial ALS, making it the most prevalent genetic mutation for a disease with an annual economic burden of $433M in the US. The expansion also appears in about 25 percent of familial FTD, as well as 7 percent of sporadic ALS and 5 percent of sporadic FTD. The C9ORF72 region is difficult to size accurately because most expansions in affected individuals are more than 700 repeats in length. Currently testing relies on "homebrew" PCR-based assays for accurate sizing of <35 repeats, or, separately, Southern blot analysis for crude sizing of >35 repeats. A solution is needed to enable high throughput, reliable, sensitive, and accurate sizing of the repeat region for both short (<35 repeats) and long expansions (>35 to 1000 repeats) in a single assay. The proposed assay offers a solution to these technical challenges based on the repeat-primed assay platform (Amplidex(R) FMR1 PCR) that Asuragen has developed and successfully commercialized for fragile X syndrome, a CGG triplet repeat disorder. We will leverage >5 years of experience in optimizing high performance diagnostic assays for GC- rich repeat sequences, including methods for the routine amplification of at least 1300 CGG repeats, to develop an accurate and robust single tube molecular diagnostic test for C9ORF72. The specific aims of this proposal are: Aim 1. Optimize the detection of C9ORF72 hexanucleotide repeat expansions using a repeat-primed PCR strategy. Aim 2. Optimize and validate a single tube repeat sizing assay using previously characterized genomic DNA samples from Coriell cell repositories. 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, to further an understanding of known and novel genotype-phenotype associations, and to enable opportunities for targeted therapeutics and clinical trials.

描述（由申请人提供）：该项目的长期目标是开发、验证和商业化一种检测方法，以改进 C9ORF72 的筛选，C9ORF72 是第 9 号染色体上的一个基因，与额颞叶痴呆 (FTD) 和肌萎缩侧索硬化症 (ALS) 相关。 C9ORF72 非编码区中富含鸟嘌呤和胞嘧啶的六核苷酸重复序列 (GGGGCC) 的扩展与高达 67% 的家族性 ALS 相关，使其成为美国每年经济负担达 4.33 亿美元的疾病最常见的基因突变。这种扩张还出现在约 25% 的家族性 FTD、7% 的散发性 ALS 和 5% 的散发性 FTD 中。 C9ORF72 区域很难准确确定大小，因为受影响个体中的大多数扩增长度都超过 700 个重复。目前的测试依赖于基于“自制”PCR 的测定来精确确定<35 个重复的大小，或者单独使用Southern blot 分析来确定>35 个重复的粗略大小。需要一种解决方案，能够在单次测定中实现短（<35 次重复）和长扩展（>35 至 1000 次重复）的重复区域的高通量、可靠、灵敏和准确的大小调整。所提出的检测方法基于 Asuragen 开发的重复引发检测平台 (Amplidex(R) FMR1 PCR) 并成功商业化用于脆性 X 综合征（一种 CGG 三联体重复疾病），为这些技术挑战提供了解决方案。我们将利用超过 5 年的优化富含 GC 重复序列的高性能诊断测定的经验，包括至少 1300 个 CGG 重复序列的常规扩增方法，来开发针对 C9ORF72 的准确且稳健的单管分子诊断测试。该提案的具体目标是： 目标 1. 使用重复引发的 PCR 策略优化 C9ORF72 六核苷酸重复扩增的检测。 目标 2. 使用来自 Coriell 细胞存储库的先前表征的基因组 DNA 样本优化并验证单管重复大小测定。 开发一种改进的、信息丰富的 C9ORF72 检测方法将有助于作为 ALS 和 FTD 的筛查和诊断测试，以及临床研究工具，以确定与其他形式的年龄发病神经变性潜在相关的中间和/或扩大的重复大小，以进一步了解已知和新颖的基因型-表型关联，并为靶向治疗和临床试验提供机会。