BACKGROUND A repeat expansion in the C9orf72-SMCR8 complex subunit (C9orf72) is the most common genetic cause of two debilitating neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Currently, much remains unknown about which variables may modify these diseases. We sought to investigate associations between C9orf72 promoter methylation, RNA expression levels, and repeat length, their potential effects on disease features, as well as changes over time and within families. METHODS All samples were obtained through the ALS Center at Mayo Clinic Florida. Our primary cohort included 75 unrelated patients with an expanded C9orf72 repeat, 33 patients who did not possess this expansion, and 20 control subjects without neurodegenerative diseases. Additionally, 67 members from 17 independent C9orf72 families were selected of whom 33 harbored this expansion. Longitudinally collected samples were available for 35 C9orf72 expansion carriers. To increase our understanding of C9orf72-related diseases, we performed quantitative methylation-sensitive restriction enzyme-based assays, digital molecular barcoding, quantitative real-time PCR, and Southern blotting. RESULTS In our primary cohort, higher methylation levels were observed in patients with a C9orf72 repeat expansion than in patients without this expansion (p = 1.7e-13) or in control subjects (p = 3.3e-07). Moreover, we discovered that an increase in methylation levels was associated with a decrease in total C9orf72 transcript levels (p = 5.5e-05). These findings aligned with our observation that C9orf72 expansion carriers had lower expression levels of total C9orf72 transcripts than patients lacking this expansion (p = 3.7e-07) or control subjects (p = 9.1e-05). We also detected an elevation of transcripts containing intron 1a (upstream of the repeat) in patients carrying a C9orf72 repeat expansion compared to (disease) controls (p ≤ 0.01), an indication of abortive transcripts and/or a switch in transcription start site usage. While methylation and expression levels were relatively stable over time, fluctuations were seen in repeat length. Interestingly, contractions occurred frequently in parent-offspring transmissions (> 50%), especially in paternal transmissions. Furthermore, smaller repeat lengths were detected in currently unaffected individuals than in affected individuals (p = 8.9e-04) and they were associated with an earlier age at collection (p = 0.008). CONCLUSIONS In blood from C9orf72 expansion carriers, we found elevated methylation levels, reduced expression levels, and unstable expansions that tend to contract in successive generations, arguing against anticipation.

中文翻译：

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在C9orf72扩增载体的临床队列中，甲基化水平升高，表达水平降低和频繁收缩。

背景技术C9orf72-SMCR8复合亚基（C9orf72）中的重复扩增是两种使人衰弱的神经退行性疾病：肌萎缩性侧索硬化（ALS）和额颞痴呆（FTD）的最常见遗传原因。目前，关于哪些变量可以改变这些疾病的知之甚少。我们试图研究C9orf72启动子甲基化，RNA表达水平和重复长度，它们对疾病特征的潜在影响以及随时间和家庭变化的关系。方法所有样品均通过佛罗里达州梅奥诊所的ALS中心获得。我们的主要队列研究包括75名无关的C9orf72重复序列扩大的患者，33例不具有这种扩大的患者以及20名无神经退行性疾病的对照组。另外，从17个独立的C9orf72家族中选择了67位成员，其中33位成员进行了这种扩展。纵向收集的样品可用于35个C9orf72扩展载体。为了增加对C9orf72相关疾病的了解，我们进行了基于甲基化敏感的限制性内切酶的定量分析，数字分子条形码，定量实时PCR和Southern印迹分析。结果在我们的主要队列中，观察到具有C9orf72重复扩增患者的甲基化水平高于没有该扩增患者（p = 1.7e-13）或对照组（p = 3.3e-07）。此外，我们发现甲基化水平的提高与总C9orf72转录水平的降低有关（p = 5.5e-05）。这些发现与我们的观察结果相符，即C9orf72扩展载体的总C9orf72转录本的表达水平低于缺乏这种扩展的患者（p = 3.7e-07）或对照组（p = 9.1e-05）。我们还检测到与（疾病）对照相比，携带C9orf72重复扩增的患者中含有内含子1a（重复序列的上游）的转录本升高（p≤0.01），表明流产的转录本和/或转录起始位点使用情况发生了变化。虽然甲基化和表达水平随时间相对稳定，但重复长度出现波动。有趣的是，在父母-子女的传播中，收缩频繁发生（> 50％），尤其是在父亲的传播中。此外，在当前未受影响的个体中检测到的重复长度比在受影响的个体中更小（p = 8）。9e-04），并且它们与收集时的较早年龄相关（p = 0.008）。结论在来自C9orf72扩增载体的血液中，我们发现甲基化水平升高，表达水平降低以及不稳定的扩增，这些扩增往往会在连续的世代中收缩，这与预期相抵触。