C9ORF72 levels: implications for diagnosis, prognosis, and treatment of ALS and related disorders

项目摘要/摘要 新的证据表明，9号染色体开放阅读框架72(C9ORF72)表达，RNA焦点， 二肽重复蛋白与C9ORF72相关疾病有关；然而，仍有许多未知之处 关于导致这些致命的神经退行性疾病的机制。在C9ORF72中重复扩展， 肌萎缩侧索硬化症(ALS)和额颞叶痴呆最常见的遗传原因是什么 (FTD)与相当大的临床变异性有关。到目前为止，已经有三个C9ORF72成绩单 报道(变异体1、变异体2和变异体3)，它们产生两种蛋白质亚型(C9-S和C9-L)。论 根据我们最近的发现和令人兴奋的初步数据，我们假设 C9ORF72转录本变异或蛋白亚型可能导致临床上的异质性 并可能成为这些致命疾病急需的生物标志物。 我们对两个脑区C9ORF72 RNA水平的评估显示，转录本显著增加 包含重复扩展(内含子1a)之前的内含子区域，但不在后续区域中 (内含子1b)，表明C9ORF72转录本的至少一个子集被截断。在小脑中，我们观察到 包含整个第一内含子的转录本和二肽重复蛋白之间的显著关联(即， Poly[GP]和Poly[GA])，表明这些转录本可能作为重复相关的非ATG的模板 (Ran)翻译。重要的是，我们还注意到C9ORF72转录本与 变异体1和发病后的存活率，在额叶皮质和小脑，这值得警惕 针对C9ORF72(例如，反义寡核苷酸[ASOS])的新治疗策略的开发。 为了解决缺乏有效的生物标记物来确定疾病预后和分期的问题，监测目标 参与和患者对潜在治疗干预的反应，我们现在提出详细的 表达研究。我们将使用Northern blotting技术评估已知的C9ORF72转录本 以及在一系列区域中存在额外的转录变体和/或异常物种，例如 小脑、额叶皮质、运动皮质、海马体、杏仁核、基底节、丘脑、延髓和脊髓 电源线。我们将对一大批有症状和症状前的人群进行数字分子条形码编码 扩展携带者和疾病控制，重点关注C9ORF72转录变异体1在 血液；然而，我们也将调查其他转录和区域(目标1)。最后，我们将评估 用Western blotting技术和敏感的免疫分析测定C9ORF72蛋白亚型的水平，我们 将使用免疫组织化学和RNA荧光原位杂交(FISH； 目标2)。在这样做的过程中，我们将确定特定的C9ORF72转录本或蛋白质亚型 可作为C9ORF72相关疾病的生物标志物。

PROJECT SUMMARY/ABSTRACT Emerging evidence suggests that chromosome 9 open reading frame 72 (C9ORF72) expression, RNA foci, and dipeptide-repeat proteins contribute to C9ORF72-related diseases; however, much remains unknown about the mechanisms causing these fatal neurodegenerative diseases. Repeat expansions in C9ORF72, which are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are associated with considerable clinical variability. To date, three C9ORF72 transcripts have been reported (variant 1, variant 2, and variant 3), which give rise to two protein isoforms (C9-S and C9-L). On the heels of our recent discoveries and exciting preliminary data, we hypothesize that the expression of C9ORF72 transcript variants or protein isoforms might contribute to the clinical heterogeneity among expansion carriers and may serve as an urgently needed biomarker for these fatal diseases. Our evaluation of C9ORF72 RNA levels in two brain regions revealed a significant increase in transcripts containing the intronic area that precedes the repeat expansion (intron 1a) but not in the succeeding area (intron 1b), indicating at least a subset of C9ORF72 transcripts is truncated. In the cerebellum, we observed a significant association between transcripts containing the entire first intron and dipeptide-repeat proteins (i.e., poly[GP] and poly[GA]), suggesting these transcripts may serve as templates for repeat-associated non-ATG (RAN) translation. Importantly, we also noticed a significant association between C9ORF72 transcript variant 1 and survival after onset, both in the frontal cortex and cerebellum, which warrants caution for the development of new treatment strategies targeting C9ORF72 (e.g., antisense oligonucleotides [ASOs]). To address the lack of validated biomarkers to determine disease prognosis and stage, monitor target engagement and patient responses to potential therapeutic interventions, we now propose detailed expression studies. We will employ Northern blotting techniques to evaluate known C9ORF72 transcripts as well as the existence of additional transcript variants and/or abnormal species in a range of regions, such as cerebellum, frontal cortex, motor cortex, hippocampus, amygdala, basal ganglia, thalamus, medulla, and spinal cord. We will perform digital molecular barcoding on a large cohort of symptomatic and presymptomatic expansion carriers and disease controls, focusing on the expression levels of C9ORF72 transcript variant 1 in blood; however, we will also investigate other transcripts and regions (Aim 1). Finally, we will evaluate the levels of C9ORF72 protein isoforms using Western blotting techniques and sensitive immunoassays, and we will visualize protein isoforms using immunohistochemistry and RNA fluorescent in situ hybridization (FISH; Aim 2). In so doing, we will determine whether specific C9ORF72 transcripts or protein isoforms contribute to the clinical heterogeneity and may serve as a biomarker for C9ORF72-related diseases.