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??). 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. Overall design: 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