C9ORF72 GGGGCC expanded repeats produce splicing dysregulation which correlates with disease severity in amyotrophic lateral sclerosis [HG-U133_Plus_2]

Objective: An intronic GGGGCC-repeat expansion of C9ORF72 is the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The mechanism of neurodegeneration is unknown, but a direct effect on RNA processing mediated by RNA foci transcribed from the repeat sequence has been proposed. Results: Gene level analysis revealed a number of differentially expressed networks and both cell types exhibited dysregulation of a network functionally enriched for genes encoding ‘RNA splicing’ proteins. There was a significant overlap of these genes with an independently generated list of GGGGCC-repeat protein binding partners. At the exon level, in lymphoblastoid cells derived from C9ORF72-ALS patients splicing consistency was lower than in lines derived from non-C9ORF72 ALS patients or controls; furthermore splicing consistency was lower in samples derived from patients with faster disease progression. Frequency of sense RNA foci showed a trend towards being higher in lymphoblastoid cells derived from patients with shorter survival, but there was no detectable correlation between disease severity and DNA expansion length. Significance: Up-regulation of genes encoding predicted binding partners of the C9ORF72 expansion is consistent with an attempted compensation for sequestration of these proteins. A number of studies have analysed changes in the transcriptome caused by C9ORF72 expansion, but to date findings have been inconsistent. As a potential explanation we suggest that dynamic sequestration of RNA processing proteins by RNA foci might lead to a loss of splicing consistency; indeed in our samples measurement of splicing consistency correlates with disease severity. Gene expression profiling utilised total RNA extracted from motor neurons derived from human ALS patients with an expansion of C9ORF72 (n=8), and controls (n=3).

研究目标：C9ORF72基因内含子区GGGGCC重复扩增是肌萎缩侧索硬化（amyotrophic lateral sclerosis, ALS）与额颞叶痴呆最常见的遗传变异类型。目前神经退行性变的具体机制尚不明确，但已有研究提出由重复序列转录形成的RNA病灶（RNA foci）介导的RNA加工（RNA processing）异常是潜在致病机制。
研究结果：基因水平分析鉴定出多个差异表达调控网络，两种细胞系均呈现出功能富集于RNA剪接（RNA splicing）蛋白编码基因的调控网络失调。上述差异基因与独立构建的GGGGCC重复序列蛋白结合伴侣列表存在显著重叠。在外显子水平，源自C9ORF72突变型ALS患者的淋巴母细胞系（lymphoblastoid cells）的剪接一致性（splicing consistency）显著低于非C9ORF72突变型ALS患者或健康对照来源的细胞系；此外，疾病进展更快的患者来源样本的剪接一致性更低。针对有义RNA病灶的频率分析显示，生存期更短的患者来源的淋巴母细胞系呈现出病灶频率升高的趋势，但未检测到疾病严重程度与DNA扩增长度之间存在显著相关性。
研究意义：编码C9ORF72扩增序列预测结合伴侣的基因上调表达，与机体对这些蛋白被隔离的现象产生代偿性反应相符。已有多项研究分析了C9ORF72扩增引发的转录组（transcriptome）变化，但目前相关研究结论尚未达成一致。本研究提出一种潜在解释：RNA病灶对RNA加工蛋白的动态隔离作用可能导致剪接一致性丧失；本研究样本中剪接一致性的检测结果确实与疾病严重程度显著相关。本次基因表达谱分析采用的总RNA提取自携带C9ORF72扩增突变的人类ALS患者来源的运动神经元（motor neurons，n=8）以及健康对照（n=3）。