Profiling non-coding RNA expression in cerebrospinal fluid of amyotrophic lateral sclerosis patients

Objective biomarkers for the fatal neurodegenerative disease amyotrophic lateral sclerosis or motor neuron disease (ALS/MND) are critical for diagnosis, drug development, clinical trials, and insight into disease pathology. Key candidates for biomarkers present in biofluids include non-coding RNA (ncRNA) transcripts including microRNA, piwi-interacting RNA and transfer RNA. To determine if the central nervous system was the source of the dysregulated ncRNA biomarkers we previously observed in serum, we sought to identify dysregulated ncRNA candidates in cerebrospinal fluid (CSF) which may provide new insight into the disease pathology. Small RNA sequencing (RNA-seq) was undertaken on CSF samples from healthy controls (<i>n</i> = 18), disease mimics (<i>n</i> = 8), and ALS patients (<i>n</i> = 40) in our Oxford Study for Biomarkers of ALS cohort, with RT-qPCR used to confirm their dysregulation. We found a range of ncRNA that were dysregulated in the RNA-seq screen, but these failed to be validated or detected in some cases using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, our previously identified serum ncRNA biomarker showed no change in CSF or correlation to serum. This study suggests the CSF may not be the source of dysregulated ncRNA in the serum and highlights the difficulty in identifying ncRNA in CSF as biomarkers for ALS.KEY MESSAGESIn this current study, we investigated the expression of non-coding RNA transcripts in the cerebrospinal fluid of ALS patients compared to healthy controls.RNA-seq identified dysregulated non-coding RNA transcripts, but these were not validated with RT-qPCR.We conclude that cerebrospinal fluid is not a suitable source of diagnostic biomarkers. In this current study, we investigated the expression of non-coding RNA transcripts in the cerebrospinal fluid of ALS patients compared to healthy controls. RNA-seq identified dysregulated non-coding RNA transcripts, but these were not validated with RT-qPCR. We conclude that cerebrospinal fluid is not a suitable source of diagnostic biomarkers.

致命性神经退行性疾病肌萎缩侧索硬化（amyotrophic lateral sclerosis, ALS）又称运动神经元病（motor neuron disease, MND），其客观生物标志物对于疾病诊断、药物研发、临床试验以及解析疾病病理机制均具有关键意义。生物流体中的关键候选生物标志物包括各类非编码RNA（non-coding RNA, ncRNA）转录本，例如微小RNA（microRNA）、piwi相互作用RNA（piwi-interacting RNA）以及转运RNA（transfer RNA）。
为明确此前在血清中观测到的失调ncRNA生物标志物是否源自中枢神经系统，我们旨在鉴定脑脊液（cerebrospinal fluid, CSF）中的失调ncRNA候选分子，以期为疾病病理机制研究提供新的视角。本研究依托牛津大学ALS生物标志物队列研究，对健康对照者（*n*=18）、疾病模拟组（*n*=8）以及ALS患者（*n*=40）的脑脊液样本开展小RNA测序（RNA-seq），并通过逆转录定量聚合酶链反应（RT-qPCR）验证其表达失调情况。
研究团队在RNA-seq筛选中发现了一批失调的ncRNA，但其中部分样本在使用RT-qPCR验证时未能得到确认，甚至无法被检测到。此外，我们此前鉴定出的血清ncRNA生物标志物在脑脊液中未出现表达变化，且与血清中的表达水平无相关性。本研究表明，脑脊液可能并非血清中失调ncRNA的来源，同时也凸显了将脑脊液中的ncRNA开发为ALS诊断生物标志物所面临的挑战。
【核心研究结论】
1. 本研究对比分析了ALS患者与健康对照者脑脊液中非编码RNA转录本的表达谱；
2. RNA-seq可鉴定出失调的非编码RNA转录本，但此类分子无法通过RT-qPCR完成验证；
3. 本研究得出结论：脑脊液不适宜作为ALS诊断生物标志物的来源。