Detection of brain-derived cell free DNA in plasma
Neuronal loss is a major pathological feature of neurodegener-ative diseases. The analysis of plasma cell-free DNA (cfDNA) is an emerging approach to track cell death events in a minimally invasive way, also from inaccessible areas of the body like brain. Previous studies showed that DNA methylation (DNAm) profiles can be used to map the tissue of origin of cfDNA and to identify molecules released from brain upon cell death. The aim of the present study is to contribute to this research field, presenting the development and validation of an assay for the detection of brain-derived cfDNA (bcfDNA).To identify CpG sites with brain-specific DNAm, we compared brain and non-brain tissues for their chromatin states profiles and genome-wide DNAm data, available in public datasets. The selected target genomic regions were experimentally validated by bisulfite sequencing on DNA extracted from 44 different autoptic tissues, including multiple brain regions. Sequencing data were analyzed to identify brain-specific epihaplotypes. The de-veloped assay was tested in plasma cfDNA from patients with immune effector cell-associated neurotoxicity syndrome (ICANS) following chimeric antigen receptor T (CAR-T) therapy.We validated 5 genomic regions with brain-specific DNAm (4 hypomethylated and 1 hypermethylated in brain). DNAm analysis of the selected genomic regions in plasma samples from CAR-T patients revealed higher levels of bcfDNA in participants with ongoing neurotoxicity syndrome.In conclusion, we developed an assay for the analysis of bcfDNA in plasma. The assay is a promising tool for the early detection of neuronal loss in neurodegenerative diseases.