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

描述（由申请人提供）：本项目的长期目标是开发、验证和商业化一种检测方法，以改善C9 ORF 72的筛选，C9 ORF 72是9号染色体上与额颞叶痴呆（FTD）和肌萎缩侧索硬化（ALS）相关的基因。C9 ORF 72非编码区富含鸟嘌呤和胞嘧啶的六核苷酸重复序列（GGGGCC）的扩增与高达67%的家族性ALS相关，使其成为美国每年经济负担为4.33亿美元的疾病中最普遍的遗传突变。这种扩张也出现在约25%的家族性FTD中，以及7%的散发性ALS和5%的散发性FTD中。C9 ORF 72区域很难准确地确定大小，因为受影响个体中的大多数扩增长度超过700个重复。目前的测试依赖于“自制”的基于PCR的测定，用于<35个重复的精确大小测定，或者单独地，用于>35个重复的粗大小测定的Southern印迹分析。需要一种解决方案来实现在单个测定中对短（<35个重复）和长扩增（>35至1000个重复）的重复区进行高通量、可靠、灵敏和准确的大小测定。基于Asuragen开发并成功商业化用于脆性X综合征（一种CGG三联体重复序列紊乱）的重复引物检测平台（Amplidex（R）FMR 1 PCR），所提出的检测方法为这些技术挑战提供了解决方案。我们将利用在优化富含GC重复序列的高性能诊断测定方面的>5年经验，包括用于至少1300个CGG重复序列的常规扩增的方法，以开发用于C9 ORF 72的准确且稳健的单管分子诊断测试。本提案的具体目标是：目标1。使用重复引物PCR策略优化C9 ORF 72六核苷酸重复扩增的检测。 目标2.使用来自Coriell细胞库的先前表征的基因组DNA样品优化和验证单管重复测定。 开发一种改进的、信息丰富的C9 ORF 72检测方法将可用作ALS和FTD的筛查和诊断试验，以及临床研究工具，以鉴定与其他形式的年龄发作性神经变性潜在相关的中间和/或扩增重复序列大小，进一步了解已知和新的基因型-表型关联，并为靶向治疗和临床试验提供机会。

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.