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Non‐invasive epigenomic molecular phenotyping of the human brain via liquid biopsy of cerebrospinal fluid and next generation sequencing

Our goal was to undertake a genome‐wide epigenomic liquid biopsy of cerebrospinal fluid (CSF) for the comprehensive analysis of cell‐free DNA (cfDNA) methylation signatures in the human central nervous system (CNS). Solution‐phase hybridization and massively parallel sequencing of bisulfite converted human DNA was employed to compare methylation signatures of cfDNA obtained from CSF with plasma. Recovery of cfDNA from CSF was relatively low (68–840 pg/mL) compared to plasma (2720–8390 pg/mL) and cfDNA fragments from CSF were approximately 20 bp shorter than their plasma‐derived counterparts. Distributions of CpG methylation signatures were significantly altered between CSF and plasma, both globally and at the level of functional elements including exons, introns, CpG islands, and shores. Sliding window analysis was used to identify differentially methylated regions. We found numerous gene/locus‐specific differences in CpG methylation between cfDNA from CSF and plasma. These loci were more frequently hypomethylated in CSF compared to plasma. Differentially methylated CpGs in CSF were identified in genes related to branching of neurites and neuronal development. Using the GTEx RNA expression database, we found clear association between tissue‐specific gene expression in the CNS and cfDNA methylation patterns in CSF. Ingenuity pathway analysis of differentially methylated regions identified an enrichment of functional pathways related to neurobiology. In conclusion, we present a genome‐wide analysis of DNA methylation in human CSF. Our methods and the resulting data demonstrate the potential of epigenomic liquid biopsy of the human CNS for molecular phenotyping of brain‐derived DNA methylation signatures.