7a52c1999e672f8bae781d350308e3ff 322580.pdf f4ee60c5d35ad95b2a124583d849b349091ce00b 322580.pdf d1077876ce9349498b5052ea2dd219d1f3973915290bffa14eb94ba83ee7415d 322580.pdf Title: Advancing Personalized Medicine in Alzheimer’s Disease: Liquid Biopsy Epigenomics Unveil APOE 4-Linked Methylation Signatures Subject: 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 4 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 4 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: Alzheimer’s disease; cell-free DNA; liquid biopsy; EPIC array; DNA methylation; APOE 4; blood Author: Mónica Macías, Juan José Alba-Linares, Blanca Acha, Idoia Blanco-Luquin, Agustín F. Fernández, Johana Álvarez-Jiménez, Amaya Urdánoz-Casado, Miren Roldan, Maitane Robles, Eneko Cabezon-Arteta, Daniel Alcolea, Javier Sánchez Ruiz de Gordoa, Jon Corroza, Carolina Cabello, María Elena Erro, Ivonne Jericó, Mario F. Fraga and Maite Mendioroz Creator: LaTeX with hyperref Producer: pdfTeX-1.40.25 CreationDate: Wed Apr 9 04:00:49 2025 CEST ModDate: Wed Apr 9 04:03:22 2025 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 26 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1966951 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ZVBSIG+EURM10 Type 1 Builtin yes yes yes 10 0 GXNUUP+EURB10 Type 1 Builtin yes yes yes 15 0 QKZKVH+VnURWPalladioL Type 1 Custom yes yes yes 20 0 KEGNDG+URWPalladioL-Roma Type 1 Custom yes yes yes 26 0 PZBOKA+URWPalladioL-Bold Type 1 Custom yes yes yes 32 0 QOQZJG+URWPalladioL-Ital Type 1 Custom yes yes yes 37 0 ZKYGAW+URWPalladioL-BoldItal Type 1 Custom yes yes yes 42 0 KHFIJR+EURM7 Type 1 Builtin yes yes yes 47 0 COGLGI+CMSY10 Type 1 Builtin yes yes yes 79 0 CTGVMV+EURB7 Type 1 Builtin yes yes yes 100 0 WBREXZ+PazoMath-Italic Type 1 Builtin yes yes yes 108 0 JEEEBO+PalatinoLinotype,Italic CID TrueType Identity-H yes yes yes 116 0 JEEEBP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 122 0 JEEECB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 125 0 JEEECC+PalatinoLinotype CID TrueType Identity-H yes yes yes 128 0 JEEECD+PalatinoLinotype TrueType WinAnsi yes yes no 134 0 JEEEKD+PalatinoLinotype TrueType MacRoman yes yes no 137 0 JEEEBP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 170 0 JEEECB+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 173 0 JEEECC+PalatinoLinotype CID TrueType Identity-H yes yes yes 176 0 JEEECD+PalatinoLinotype TrueType WinAnsi yes yes no 182 0 JEEEKD+PalatinoLinotype TrueType MacRoman yes yes no 185 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2025-11-27 04:04:52 CET RepresentationInformation: 322580.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2025-11-26 13:46:19 CET Size: 1966951 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 519 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Results Destination: section.2 Children: Item: Title: Characterization of Subjects and Samples Destination: subsection.2.1 Item: Title: cfDNA Concentration and Quality Destination: subsection.2.2 Item: Title: Surrogate Variable Analysis Destination: subsection.2.3 Item: Title: Differential Methylated Positions Destination: subsection.2.4 Item: Title: Correlation with AD Clinical Parameters and Biomarkers Destination: subsection.2.5 Item: Title: Functional in Silico Analysis of DMPs Destination: subsection.2.6 Item: Title: Differential Methylated Positions According to APOE 4 Status Destination: subsection.2.7 Item: Title: Differential Methylated Regions Destination: subsection.2.8 Item: Title: Orthogonal Validation Destination: subsection.2.9 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Study Design Destination: subsection.4.1 Item: Title: Subjects’ Characterization Destination: subsection.4.2 Item: Title: Blood and Cerebrospinal Fluid (CSF) Samples Destination: subsection.4.3 Item: Title: cfDNA Isolation and Quantification Destination: subsection.4.4 Item: Title: Characterization of cfDNA: Fragment Size Analysis Destination: subsection.4.5 Item: Title: Genome-Wide cfDNA Methylation Analysis Destination: subsection.4.6 Item: Title: Array Data Preprocessing Destination: subsection.4.7 Item: Title: cfDNA Cell-Type Deconvolution Destination: subsection.4.8 Item: Title: Surrogate Variable Analysis Destination: subsection.4.9 Item: Title: Probe-Level Differential Methylation Analyses Destination: subsection.4.10 Item: Title: Region-Level Differential Methylation Analyses Destination: subsection.4.11 Item: Title: Probe Annotation Destination: subsection.4.12 Item: Title: Functional In Silico Analysis of DMPs Destination: subsection.4.13 Item: Title: Orthogonal Validation Destination: subsection.4.14 Item: Title: Conclusions Destination: section.5 Item: Title: References Destination: appendix.A. Info: Title: Advancing Personalized Medicine in Alzheimer’s Disease: Liquid Biopsy Epigenomics Unveil APOE 4-Linked Methylation Signatures Author: Mónica Macías, Juan José Alba-Linares, Blanca Acha, Idoia Blanco-Luquin, Agustín F. Fernández, Johana Álvarez-Jiménez, Amaya Urdánoz-Casado, Miren Roldan, Maitane Robles, Eneko Cabezon-Arteta, Daniel Alcolea, Javier Sánchez Ruiz de Gordoa, Jon Corroza, Carolina Cabello, María Elena Erro, Ivonne Jericó, Mario F. Fraga and Maite Mendioroz Subject: 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 4 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 4 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. 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