The discovery of new biomarkers would be very valuable to improve the detection of early Alzheimer’s disease (AD). DNA methylation marks may serve as epigenetic biomarkers of early AD. Here we identified epigenetic marks that are present in the human hippocampus from the earliest stages of AD. A previous methylome dataset of the human AD hippocampus was used to select a set of 8 differentially methylated positions (DMPs) since early AD stages. Next, bisulfite pyrosequencing was performed in an expanded homogeneous cohort of 18 pure controls and 35 hippocampal samples with neuropathological changes of pure AD. Correlation between DNA methylation levels in DMPs and phospho-tau protein burden assessed by immunohistochemistry in the hippocampus was also determined. We found 4 DMPs showing higher levels of DNA methylation at early AD stages compared to controls, involving ELOVL2, GIT1/TP53I13 and the histone gene locus at chromosome 6. DNA methylation levels assessed by bisulfite pyrosequencing correlated with phospho-tau protein burden for ELOVL2 and HIST1H3E/HIST1H3F genes. In this discovery study a set of 4 epigenetic marks of early AD stages have been identified in the human hippocampus. These epigenetic changes point to specific pathways that might be involved in AD pathogenesis, such as the long-chain fatty acid biogenesis or histone functions. It would be worth studying in depth the specific pathways related to these epigenetic marks. These early alterations in DNA methylation in the AD hippocampus could be regarded as candidate biomarkers to be explored in future translational studies.

Background and Objectives There is an urgent need to identify novel noninvasive biomarkers for Alzheimer disease (AD) diagnosis. Recent advances in blood-based measurements of phosphorylated tau (pTau) species are promising but still insufficient to address clinical needs. Epigenetics has been shown to be helpful to better understand AD pathogenesis. Epigenetic biomarkers have been successfully implemented in other medical disciplines, such as oncology. The objective of this study was to explore the diagnostic accuracy of a blood-based DNA methylation marker panel as a noninvasive tool to identify patients with late-onset Alzheimer compared with age-matched controls. Methods A case-control study was performed. Blood DNA methylation levels at 46 cytosine-guanine sites (21 genes selected after a comprehensive literature search) were measured by bisulfite pyrosequencing in patients with “probable AD dementia” following National Institute on Aging and the Alzheimer9s Association guidelines (2011) and age-matched and sex-matched controls recruited at Neurology Department-University Hospital of Navarre, Spain, selected by convenience sampling. Plasma pTau181 levels were determined by Simoa technology. Multivariable logistic regression analysis was performed to explore the optimal model to discriminate patients with AD from controls. Furthermore, we performed a stratified analysis by sex. Results The final study cohort consisted of 80 patients with AD (age: median [interquartile range] 79 [11] years; 58.8% female) and 100 cognitively healthy controls (age 77 [10] years; 58% female). A panel including DNA methylation levels at NXN, ABCA7, and HOXA3 genes and plasma pTau181 significantly improved (area under the receiver operating characteristic curve 0.93, 95% CI 0.89–0.97) the diagnostic performance of a single pTau181-based model, adjusted for age, sex, and APOE ɛ4 genotype. The sensitivity and specificity of this panel were 83.30% and 90.00%, respectively. After sex-stratified analysis, HOXA3 DNA methylation levels showed consistent association with AD. Discussion These results highlight the potential translational value of blood-based DNA methylation biomarkers for noninvasive diagnosis of AD. Registration Information Research Ethics Committee of the University Hospital of Navarre (PI17/02218).

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.