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

项目总结/摘要 新出现的证据表明，9号染色体开放阅读框72（C9 ORF 72）表达，RNA灶， 和二肽重复蛋白有助于C9 ORF 72相关的疾病;然而，许多仍然未知 这些致命的神经退行性疾病的机制在C9 ORF 72中重复扩增， 这是肌萎缩侧索硬化症（ALS）和额颞叶痴呆症最常见的遗传原因 (FTD)与相当大的临床变异性有关。到目前为止，三个C9 ORF 72转录本已经被 报道了两种蛋白质异构体（变体1、变体2和变体3），其产生两种蛋白质异构体（C9-S和C9-L）。上 根据我们最近的发现和令人兴奋的初步数据，我们假设， C9 ORF 72转录变体或蛋白质同种型可能导致临床异质性， 扩展载体，并可能作为这些致命疾病急需的生物标志物。 我们对两个脑区C9 ORF 72 RNA水平的评估显示，转录本的显著增加， 包含重复扩增之前的内含子区域（内含子1a），但不在随后的区域中 （内含子1b），表明至少C9 ORF 72转录物的一个子集被截短。在小脑中，我们观察到 含有整个第一内含子的转录物和二肽重复蛋白之间的显著关联（即， poly[GP]和poly[GA]），表明这些转录本可以作为重复相关非ATG的模板 (RAN)翻译.重要的是，我们还注意到C9 ORF 72转录本与C9 ORF 72转录本之间的显著相关性。 变异1和发病后的生存率，无论是在额叶皮层和小脑，这值得谨慎， 靶向C9 ORF 72的新治疗策略的开发（例如，反义寡核苷酸[ASO]）。 为了解决缺乏经验证的生物标志物来确定疾病预后和分期的问题， 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。我们将采用北方印迹技术来评估已知的C9 ORF 72转录物， 以及在一系列区域中存在额外的转录变体和/或异常种类，例如 小脑、额叶皮质、运动皮质、海马、杏仁核、基底神经节、丘脑、髓质和脊髓 线.我们将对一个大的有症状和症状前的队列进行数字分子条形码， 扩增载体和疾病对照，集中于C9 ORF 72转录变体1在扩增载体中的表达水平。 血液;然而，我们也将研究其他转录本和区域（目的1）。最后，我们将评估 C9 ORF 72蛋白亚型的水平，使用蛋白质印迹技术和敏感的免疫测定，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，我们将确定是否特定的C9 ORF 72转录本或蛋白质亚型， 有助于临床异质性，并可作为C9 ORF 72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.