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DNA Methylation Description of Hippocampus, Cortex, Amygdala, and Blood of Drug-Resistant Temporal Lobe Epilepsy

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Abstract

Epigenetic changes such as DNA methylation were observed in drug-resistant temporal lobe epilepsy (DR-TLE), a disease that affects 25–30% of epilepsy patients. The main objective is to simultaneously describe DNA methylation patterns associated with DR-TLE in hippocampus, amygdala, surrounding cortex to the epileptogenic zone (SCEZ), and peripheral blood. An Illumina Infinium MethylationEPIC BeadChip array was performed in 19 DR-TLE patients and 10 postmortem non-epileptic controls. Overall, 32, 59, and 3210 differentially methylated probes (DMPs) were associated with DR-TLE in the hippocampus, amygdala, and SCEZ, respectively. These DMP-affected genes were involved in neurotrophic and calcium signaling in the hippocampus and voltage-gated channels in SCEZ, among others. One of the hippocampus DMPs (cg26834418 (CHORDC1)) showed a strong blood–brain correlation with BECon and IMAGE-CpG, suggesting that it could be a potential surrogate peripheral biomarker of DR-TLE. Moreover, in three of the top SCEZ’s DMPs (SHANK3, SBF1, and MCF2L), methylation status was verified with methylation-specific qPCR. The differentially methylated CpGs were classified in DMRs: 2 in the hippocampus, 12 in the amygdala, and 531 in the SCEZ. We identified genes that had not been associated to DR-TLE so far such as TBX5, EXOC7, and WRHN. The area with more DMPs associated with DR-TLE was the SCEZ, some of them related to voltage-gated channels. The DMPs found in the amygdala were involved in inflammatory processes. We also found a potential surrogate peripheral biomarker of DR-TLE. Thus, these results provide new insights into epigenetic modifications involved in DR-TLE.

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Data Availability

All data produced in the present study are available upon reasonable request to the authors.

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Acknowledgements

We are particularly grateful for the generous contribution of the patients and the collaboration of Biobanco-HUP (Hospital Universitario de la Princesa) and Biobank Network of the Region of Murcia, BIOBANC-MUR, registered on the Registro Nacional de Biobancos with registration number B.0000859. BIOBANC-MUR is supported by the “Instituto de Salud Carlos III (proyecto PT20/00109), by “Instituto Murciano de Investigación Biosanitaria Virgen de la Arrixaca, IMIB” and by “Consejeria de Salud de la Comunidad Autónoma de la Región de Murcia”. We would like to thank Manuel Gómez Gutierrez for his help with the study and their valuable comments on this manuscript. The genotyping was performed at the Spanish National Cancer Research Centre, in the Human Genotyping lab, a member of CeGen, PRB3 and is supported by grant PT17/0019, of the PE I+D+i 2013-2016, funded by ISCIII and ERDF. We would like to thank Dr. Agustín Fernández-Fernández and Hortensia de la Fuente for their valuable advice.

Funding

This study was supported by Instituto de Salud Carlos III: PI2017/02244. PSJ is funded by Industrial PhD grant from ‘Consejeria de Educación e Investigación’ of ‘Comunidad de Madrid’ developed in NIMGenetics and in Hospital Universitario de La Princesa (CAM.IND2017/BMD-7578).

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Author notes

  1. Cristina Virginia Torres-Díaz and María C. Ovejero-Benito contributed equally to the manuscript.

Authors and Affiliations

  1. Department of Clinical Pharmacology, Universidad Autónoma de Madrid, Hospital Universitario de La Princesa, Instituto de Investigaciones Sanitarias La Princesa (IIS-Princesa), Madrid, Spain

    Patricia Sánchez-Jiménez, Marcos Elizalde-Horcada, Francisco Abad-Santos & María C. Ovejero-Benito

  2. NIMGenetics Genómica y Medicina S.L., Madrid, Spain

    Patricia Sánchez-Jiménez

  3. Unidad de Bioinformática, Plataforma de Apoyo a La Investigación, Instituto de Investigaciones Sanitarias Pere Virgili (ISS-PV), Tarragona, Spain

    Marcos Elizalde-Horcada

  4. Data Analysis Unit, Hospital Universitario de La Princesa, Instituto de Investigación Sanitaria La Princesa (IIS-IP), Madrid, Spain

    Ancor Sanz-García

  5. Department of Clinical Analysis, Hospital Universitario de La Princesa, Madrid, Spain

    Inmaculada Granero-Cremades

  6. Department of Neurology, Hospital Universitario de La Princesa, Madrid, Spain

    María De Toledo & Ana Beatriz Gago-Veiga

  7. Department of Neurosurgery, Hospital Universitario de La Princesa, Madrid, Spain

    Paloma Pulido, Marta Navas & Cristina Virginia Torres-Díaz

  8. Genetic & Molecular Epidemiology Group, Spanish National Cancer Research Center (CNIO), Madrid, Spain

    Lola Alonso-Guirado

  9. Department of Clinical Genetics, Hospital Universitario de La Princesa, Madrid, Spain

    María Concepción Alonso-Cerezo & Desirée Nava-Cedeño

  10. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain

    Francisco Abad-Santos

  11. Departamento de Ciencias Farmacéuticas y de La Salud, Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización Montepríncipe, 28660 Boadilla del Monte, Madrid, Spain

    María C. Ovejero-Benito

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  1. Patricia Sánchez-Jiménez
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Contributions

PSJ: Data curation, formal analysis, methodology, validation, and visualization. MEH and ASC: Data curation, formal analysis, methodology, and visualization. IGC: Investigation and resources. MdT, PP, MN, ABGV, MCAC, DNC, and FAS: Resources. LAG: Visualization and methodology. CVTD: Resources, project administration, funding acquisition. MCOB: Conceptualization, formal analysis, data curation, methodology, investigation, visualization, project administration, funding acquisition, supervision, and writing—original draft. All authors have read, reviewed, and approved the final manuscript.

Corresponding author

Correspondence to María C. Ovejero-Benito.

Ethics declarations

Ethical Approval

The protocol and the Informed Consent Form were approved by the Independent Clinical Research Ethics Committee of the Hospital Universitario de La Princesa. The study followed the STROBE guidelines and the Revised Declaration of Helsinki.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

Consent to Publish

This manuscript does not contain any individual person’s data in any form (including any individual details, images, or videos).

Conflict of Interest

F Abad-Santos has been a consultant or investigator in clinical trials sponsored by the following pharmaceutical companies: Abbott, Alter, Chemo, Farmalíder, Ferrer, GlaxoSmithKline, Gilead, Janssen-Cilag, Kern, Normon, Novartis, Servier, Teva, and Zambon. AB Gago-Veiga has received honoraria as a consultant and speaker for: AbbVie-Allergan, Chiesi, Exeltis, Novartis, Eli Lilly, and Teva. MC Ovejero-Benito has potential conflicts of interest (honoraria for speaking and research support) with Janssen-Cilag and Leo Pharma. The rest of the authors have no relevant financial or non-financial interests to disclose.

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Supplementary Information

Below is the link to the electronic supplementary material.

12035_2022_3180_MOESM1_ESM.jpg

Supplementary Fig. 1 Enrichment analysis performed with FUMA GWAS analyzing functions of the genes involved in DMPs in Cortical surrounding zone of patients compared with cortex of healthy controls. A) Biocarta. B) KEGG pathways. Red bars represent the proportion of overlapping genes in gene set. Blue bars show the enrichment p value, represented as the -logarithm of the FDR adjusted p value. Orange squares show the genes involved in every enrichment term. Abbreviation: FDR: false discovery rate. (JPG 902 KB)

12035_2022_3180_MOESM2_ESM.jpg

Supplementary Fig. 2 A) Metrics of the brain-blood correlation of the 32 differentially methylated probes observed in the hippocampus by BECon. B) Inter-individual variability of cg26834418 identified previously in blood-based studies of psychiatric disorders by BECon. C) Blood brain correlation of cg26834418 observed with IMAGE-CpG. (JPG 740 KB)

12035_2022_3180_MOESM3_ESM.jpg

Supplementary Fig. 3 An example of a differentially methylated region of the amygdala. Upper panel depicts coordinates in chromosome 3 (hg19). Orange squares represent the genes located in the chromosomic region shown. Green vertical lines show probes in the EPIC array. Differentially methylated region is shown in purple. Then, methylation values are shown for every control (green) or every patient (orange). Methylation values of every sample are shown in red or blue. Bottom panel shows methylation beta values, smoothed lines denote mean methylation levels for controls (C, forest green) and patients (T, orange). Each point represents the methylation level of a particular individual at a specific genomic location. (JPG 461 KB)

12035_2022_3180_MOESM4_ESM.jpg

Supplementary Fig. 4 An example of a differentially methylated region of the surrounding cortex to the epileptogenic zone. Upper panel depicts coordinates in chromosome 3 (hg19). Orange squares represent the genes located in the chromosomic region shown. Green vertical lines show probes in the EPIC array. Differentially methylated region is shown in purple. Then, methylation values are shown for every control (green) or every patient (orange). Methylation values of every sample are shown in red or blue. Bottom panel shows methylation beta values, smoothed lines denote mean methylation levels for controls (C, forest green) and patients (T, orange). Each point represents the methylation level of a particular individual at a specific genomic location. (JPG 487 KB)

12035_2022_3180_MOESM5_ESM.jpg

Supplementary Fig. 5 Correlation between drug resistant patients’ real age and predicted age by the epigenetic clock in the different tissues. A) Hippocampus, B) Amygdala, C) Surrounding cortex to the epileptogenic zone, D) Peripheral blood after adjusting by the different cell types. *p<0.05 (JPG 206 KB)

Supplementary Table 1. Sequence of primers and probes designed for methylation-specific qPCR. (XLSX 11.1 KB)

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Supplementary Table 2. Significant differentially methylated probes associated with drug resistant epilepsy in different tissues. A) Hippocampus, B) Amygdala, C) Surrounding cortex to the epileptogenic zone. Probe location and the gene annotation were taken from Illumina reference files. * Body: Gene body; TSS1500: 1500 bp upstream of transcriptional start site (TSS): TSS200, 200bp upstream of TSS; UTR: untranslated region. %Δβ: Percentage of methylation differences between the drug resistant temporal lobe epilepsy patients and controls. chr: chromosome; FDR: false discovery rate. Probes hypomethylated in patients with respect to controls are shown in green. Probes hypermethylated in patients with respect to controls are shown in red. (XLSX 281 KB)

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Supplementary Table 3. Enrichr of the significant differentially methylated probes associated with drug resistant temporal lobe epilepsy in different tissues. A) Hippocampus, B) Amygdala, C) Surrounding cortex to the epileptogenic zone. (XLSX 67.1 KB)

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Supplementary Table 4. Definition of the main clusters created by Cytoscape. Cluster 1 includes 81 proteins involved in functions such as actin filament organization, growth factors and kinases. Cluster 2 is composed by 23 proteins Voltage-gated channel, and Transient receptor potential channels. Cluster 3 is formed by 20 proteins related to the spliceosome. Cluster 4 has 16 proteins involved in DNA repair. Proteins included in cluster 1 are shown in orange, cluster 2 in blue, cluster 3 in green and cluster 4 in yellow. (XLSX 174 KB)

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Supplementary Table 5. Significant differentially methylated regions found in the different tissues (A-C). A) Hippocampus, B) Amygdala, C) Surrounding cortex to the epileptogenic zone. D) Enrichr of the genes located on the differentially methylated regions located in the surrounding cortex to the epileptogenic zone. Abbreviations: chr: chromosome; HMFDR: harmonic mean of the individual; meandiff: Mean differences in DNA methylation (%) between patients and controls are shown as a measurement of the effect size. Fisher <0.05 is considered significant. (XLSX 80 KB)

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Supplementary Table 6. A) Number of samples from the different tissues available for the different clinical and demographic factors studied. B) Significant differentially methylated probes associated with structural etiology in different tissues. * Body: Gene body; TSS1500: 1500 bp upstream of transcriptional start site (TSS): TSS200, 200bp upstream of TSS; UTR: untranslated region. %Δβ: Percentage of methylation differences between the drug resistant epilepsy patients and controls. chr: chromosome; FDR: false discovery rate. (XLSX 13 KB)

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Sánchez-Jiménez, P., Elizalde-Horcada, M., Sanz-García, A. et al. DNA Methylation Description of Hippocampus, Cortex, Amygdala, and Blood of Drug-Resistant Temporal Lobe Epilepsy. Mol Neurobiol 60, 2070–2085 (2023). https://doi.org/10.1007/s12035-022-03180-z

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