Epigenetic profiles of tissue informative CpGs inform ALS disease status and progression

Background: Cell-free DNA (cfDNA), derived from dying cells, has demonstrated utility across multiple clinical applications. However, its potential in neurodegenerative diseases remains underexplored, with most existing cfDNA technologies tailored to specific disease contexts like cancer or non-invasive prenatal screening. Methods: To address this gap, we developed a novel approach to characterize epigenetic cfDNA profiles by identifying key regions of DNA methylation that reveal the tissues origins undergoing apoptosis or necrosis. We evaluated this method in the largest cfDNA study of amyotrophic lateral sclerosis (ALS) and other neurological diseases (OND) to date, encompassing two independent cohorts (n=192) from Australia (UQ Ncases=48, Ncontrols=32, NOND=15) and the USA, (UCSF Ncases=50 , Ncontrols=45)).Results: Our approach accurately distinguished ALS patients from controls (UQ AUC=0.82, UCSF AUC=0.99) and from individuals with other neurological diseases (AUC=0.91). It also identified an asymptomatic carrier of a pathogenic C9orf72 variant, and strongly correlated with ALS disease progression measures (Pearson’s R=0.66, p=3.71×10⁻⁹). Conclusions: We identified DNA methylation signals from multiple tissue types in ALS cfDNA, highlighting diverse tissue involvement in ALS pathology. These findings promote epigenetic cfDNA analysis as a powerful tool for advancing our understanding of neurodegenerative disease. Cell-free DNA was extracted from 46 Controls and 50 ALS patients from the UCSF, and 48 controls and 48 ALS patients from the University of Queensland. Cell free DNA was extracted from 20mL of whole blood in control patients and 10mL of whole blood for cases. cfDNA was then bisulfite converted and underwent high throughput methylation sequencing

背景：无细胞DNA（Cell-free DNA, cfDNA）源自死亡细胞，已在多种临床场景中展现出应用价值。然而，其在神经退行性疾病中的应用潜力仍未得到充分挖掘，当前多数已有的cfDNA技术均针对特定疾病场景开发，例如癌症或无创产前筛查。
方法：为填补这一研究空白，我们开发了一种全新的表观遗传cfDNA特征分析方法，通过识别关键DNA甲基化区域，以揭示发生细胞凋亡或坏死的组织来源。我们将该方法应用于迄今为止规模最大的肌萎缩侧索硬化症（Amyotrophic Lateral Sclerosis, ALS）及其他神经系统疾病（Other Neurological Diseases, OND）cfDNA研究中，纳入了来自澳大利亚和美国的两个独立队列（总样本量n=192）：其中澳大利亚昆士兰大学（UQ）队列包含48例ALS患者、32例健康对照及15例其他神经系统疾病患者；美国加州大学旧金山分校（UCSF）队列包含50例ALS患者及45例健康对照。
结果：本研究开发的方法可精准区分ALS患者与健康对照（昆士兰大学队列受试者工作特征曲线下面积（AUC）=0.82，加州大学旧金山分校队列AUC=0.99），同时也能精准区分ALS患者与其他神经系统疾病患者（AUC=0.91）。该方法还成功识别出1名携带致病性C9orf72变异的无症状携带者，且与ALS疾病进展指标呈现显著相关性（皮尔逊相关系数R=0.66，p=3.71×10⁻⁹）。
结论：我们在ALS患者的cfDNA中检测到来自多种组织类型的DNA甲基化信号，提示ALS病理过程中存在多组织受累的情况。本研究结果表明，表观遗传cfDNA分析可作为一种强有力的工具，助力我们加深对神经退行性疾病的理解。
实验操作细节：我们从加州大学旧金山分校队列的46名健康对照与50名ALS患者、昆士兰大学队列的48名健康对照与48名ALS患者中提取无细胞DNA。其中健康对照个体采集20mL全血，ALS患者采集10mL全血。提取得到的cfDNA经亚硫酸氢盐转化后，进行高通量甲基化测序。