Advancing Personalized Medicine in Alzheimer's Disease: Liquid Biopsy Epigenomics Unveil APOE ε4-Linked Methylation Signatures

Abstract

Recent studies show that patients with Alzheimer's disease (AD) harbor specific methylation marks in the brain that, if accessible, could be used as epigenetic biomarkers. Liquid biopsy enables the study of circulating cell-free DNA (cfDNA) fragments originated from dead cells, including neurons affected by neurodegenerative processes. Here, we isolated and epigenetically characterized plasma cfDNA from 35 patients with AD and 35 cognitively healthy controls by using the Infinium^® MethylationEPIC BeadChip array. Bioinformatics analysis was performed to identify differential methylation positions (DMPs) and regions (DMRs), including APOE ε4 genotype stratified analysis. Plasma pTau181 (Simoa) and cerebrospinal fluid (CSF) core biomarkers (Fujirebio) were also measured and correlated with differential methylation marks. Validation was performed with bisulfite pyrosequencing and bisulfite cloning sequencing. Epigenome-wide cfDNA analysis identified 102 DMPs associated with AD status. Most DMPs correlated with clinical cognitive and functional tests including 60% for Mini-Mental State Examination (MMSE) and 80% for Global Deterioration Scale (GDS), and with AD blood and CSF biomarkers. In silico functional analysis connected 30 DMPs to neurological processes, identifying key regulators such as SPTBN4 and APOE genes. Several DMRs were annotated to genes previously reported to harbor epigenetic brain changes in AD (HKR1, ZNF154, HOXA5, TRIM40, ATG16L2, ADAMST2) and were linked to APOE ε4 genotypes. Notably, a DMR in the HKR1 gene, previously shown to be hypermethylated in the AD hippocampus, was validated in cfDNA from an orthogonal perspective. These results support the feasibility of studying cfDNA to identify potential epigenetic biomarkers in AD. Thus, liquid biopsy could improve non-invasive AD diagnosis and aid personalized medicine by detecting epigenetic brain markers in blood.

Keywords: APOE ε4; Alzheimer’s disease; DNA methylation; EPIC array; blood; cell-free DNA; liquid biopsy.

Figure 1

Gene structure distribution of DMPs between patients with Alzheimer’s disease (AD) and controls. The bar chart shows results for the log2 ratios of observed (fraction of differentially methylated probes overlapping a given region) to expected (fraction of probes selected for analysis overlapping a given region) for a genomic region. Dark green boxes represent a significant enrichment (p-value < 0.05) for a particular feature. TSS = number of nucleotides upstream and downstream of the transcription start site.

Figure 2

APP gene plays a central role in the principal network evolving our AD-related DMPs. The graph shows how amyloid precursor protein-encoding gene (APP) acts as a core regulator of 12 genes found in our dataset (IPA score = 23). Red and green coloring indicate increased/decreased measurement in our dataset, respectively. Orange and blue coloring indicate predicted activation/inhibition genes participating. The intensity of the color refers to the strength of the effect.

Figure 3

Differential methylated region (DMR) annotated to HKR1 gene in plasma cfDNA from Alzheimer’s disease (AD) and control subjects. (a) The graph depicts the genomic location of the amplicon covering the DMR within the HKR1 gene’s promoter region analyzed by bisulfite cloning sequencing. Functional elements predicted for nine human cell lines, identified through chromatin immunoprecipitation (ChIP) combined with massively parallel DNA sequencing, are displayed in the middle of the graph. The track was obtained from chromatin state segmentation by HMM from ENCODE/Broad track, shown in the UCSC Genome Browser. At the bottom, the CpG island is represented by a green box, the DMR by an orange box, and the amplicon spanning the DMR is represented in yellow. (b) HKR1 cfDNA methylation levels measured by pyrosequencing. Box plot charts display the methylation levels for individual CpG sites within the amplicon and the average levels between patients with Alzheimer’s disease (AD) and controls. Horizontal lines represent median methylation values and interquartile range for each group. * p-value < 0.05; *** p-value < 0.001 (Student’s t test). (c) Representative examples of bisulfite cloning sequencing validation for the amplicon containing the CpG are shown. Black and white circles denote methylated and unmethylated cytosines, respectively. Each column symbolizes a unique CpG site in the examined amplicon and each line represents an individual DNA clone. CpG—cytosine guanine dinucleotide.