Alzheimer’s disease (AD) is the most common neurodegenerative disease affecting the elderly, with its diagnosis at early stages crucial for effective intervention. Recent evidence increasingly supports the role of epigenetic alterations in AD pathogenesis, highlighting the need for innovative biomarkers that reflect these changes. This study aimed to characterize the genome-wide DNA methylation profiles of cell-free DNA in peripheral blood for potential biomarkers associated with AD.

The Illumina Infinium array was utilized to detect the methylation patterns of circulating cell-free DNA from AD patients and healthy controls. The R Bioconductor Linear Models for Microarray Data (LIMMA) package was employed to identify methylation variable positions (MVPs), and Probe Lasso was used to pinpoint differentially methylated regions (DMRs) linked to AD. Bioinformatics enrichment analysis of the annotated genes was performed using EnrichR. A second cohort was recruited to validate the methylation changes at the C-terminal binding protein1 (CTBP1) promoter cytosine-phosphate-guanine (CpG) sites via pyrosequencing. Additionally, microarray data from the Gene Expression Omnibus (GEO) database were analyzed to further validate gene expression and immune infiltration.

A unique DNA methylation landscape in peripheral blood was characterized for AD patients and 4335 MVPs showed significant differential methylation (p < 0.01). Functional annotation and pathway enrichment analysis underscored processes and pathways inherent in the nervous system. Probe Lasso identified 68 DMRs annotated to 10 genes, with hypermethylation of CpG islands in the CTBP1 TSS1500 promoter showing significant differences when AD and controls were compared (p < 0.01), with an area under the receiver operating characteristic (ROC) curve (AUC) of 0.779. Analysis of immune cell infiltration revealed CTBP1 expression is significantly correlated with altered distribution of immune cells (p < 0.001), underscoring its potential role in modulating immune responses in AD. Moreover, CTBP1 expression levels significantly varied across multiple GEO datasets.

AD displays distinct DNA methylation patterns in peripheral blood and CTBP1 promoter hypermethylation represents a promising potential biomarker for AD diagnosis.