MINI REVIEW published: 24 March 2022 doi: 10.3389/fncel.2022.852151 The Potential of Circulating Cell-Free DNA Methylation as an Epilepsy Biomarker Ricardo Martins-Ferreira 1,2,3,4 , Bárbara Guerra Leal 2,3,4 * and Paulo Pinho Costa 2,3,4,5 1 Epigenetics and Immune Disease Group, Josep Carreras Research Institute (IJC), Barcelona, Spain, 2 Immunogenetics Lab, Molecular Pathology and Immunology, Instituto de Ciências Biomédicas Abel Salazar—Universidade do Porto (ICBAS-UPorto), Porto, Portugal, 3 Autoimmunity and Neuroscience Group, Unit for Multidisciplinary Research in Biomedicine (UMIB), ICBAS-UPorto, Porto, Portugal, 4 Laboratory for Integrative and Translational Research in Population Health (ITR), Porto, Portugal, 5 Instituto Nacional de Saúde Dr. Ricardo Jorge, Department of Human Genetics, Porto, Portugal Circulating cell-free DNA (cfDNA) are highly degraded DNA fragments shed into the bloodstream. Apoptosis is likely to be the main source of cfDNA due to the matching sizes of cfDNA and apoptotic DNA cleavage fragments. The study of cfDNA in liquid biopsies has served clinical research greatly. Genetic analysis of these circulating fragments has been used in non-invasive prenatal testing, detection of graft rejection in organ transplants, and cancer detection and monitoring. cfDNA sequencing is, however, of limited value in settings in which genetic association is not well-established, such as most neurodegenerative diseases.Recent studies have taken advantage of the cell-type Edited by: Diana Cunha-Reis, specificity of DNA methylation to determine the tissue of origin, thus detecting ongoing University of Lisbon, Portugal cell death taking place in specific body compartments. Such an approach is yet to be Reviewed by: developed in the context of epilepsy research. In this article, we review the different Ana Laura Márquez-Aguirre, CONACYT Centro de Investigación y approaches that have been used to monitor cell-type specific death through DNA Asistencia en Tecnología y Diseño del methylation analysis, and recent data detecting neuronal death in neuropathological Estado de Jalisco (CIATEJ), Mexico settings. We focus on the potential relevance of these tools in focal epilepsies, like *Correspondence: Mesial Temporal Lobe Epilepsy with Hippocampal Sclerosis (MTLE-HS), characterized Bárbara Guerra Leal baguerraleal@gmail.com by severe neuronal loss. We speculate on the potential relevance of cfDNA methylation screening for the detection of neuronal cell death in individuals with high risk of Specialty section: This article was submitted to epileptogenesis that would benefit from early diagnosis and consequent early treatment. Cellular Neuropathology, a section of the journal Keywords: cell-free DNA, DNA methylation, epilepsy, epileptogenesis, MTLE-HS, biomarker Frontiers in Cellular Neuroscience Received: 10 January 2022 Accepted: 24 February 2022 INTRODUCTION Published: 24 March 2022 The search for effective biomarkers is one of the most challenging tasks in epilepsy research. Citation: Epileptogenesis is now considered a progressive process, with seizure activity contributing to Martins-Ferreira R, Leal BG and Costa PP (2022) The Potential of extensive damage and seizure recurrence (Pitkänen et al., 2015). Epilepsy diagnosis must thus be Circulating Cell-Free DNA Methylation viewed as a race against the clock, aiming for the detection, as early as possible, of individuals as an Epilepsy Biomarker. Front. Cell. Neurosci. 16:852151. Abbreviations: cfDNA, cell-free DNA; DMP, differentially methylated position; DMR, differentially methylated region; doi: 10.3389/fncel.2022.852151 HS, hippocampal sclerosis; MTLE, mesial temporal lobe epilepsy. Frontiers in Cellular Neuroscience | www.frontiersin.org 1 March 2022 | Volume 16 | Article 852151 Martins-Ferreira et al. Cell-Free DNA Methylation in Epilepsy with a high risk of epilepsy development, that could be prenatal testing, is used to detect chromosomal abnormalities candidates for preventive pharmacological intervention. The (Fan et al., 2012). Transplant graft rejection is also monitored by search for novel viable biomarkers is considered crucial to the detection of donor-derived cfDNA sequences (De Vlaminck improve the current hurdles to early diagnosis and prognostic et al., 2014). Throughout the last two decades, cfDNA sequencing monitoring in epileptic patients. A major issue continues to be has been increasingly applied to cancer detection (Chabon the high refractoriness rates (estimated at 30%) across different et al., 2020) and also in post-therapy prognosis (Nabet et al., types of epilepsy (Pitkänen et al., 2016). Epilepsy treatment 2020; Powles et al., 2021). These liquid biopsies provide for is based on the control of ictogenesis. Current anti-seizure non-invasive detection of tumor DNA in biological fluids, also drugs target seizure control and are not directed to the whole denominated as circulating tumor DNA. epileptogenic landscape. Additionally, such biomarkers would The relevance and potential of cfDNA does not end in its facilitate economically feasible clinical trials for the development genetics, though. The utility of nucleotide sequencing is narrow of new and more effective anti-epileptic therapies by allowing a in non-mutation rich pathologies, meaning conditions in which more precise categorization of individuals with high probability a clear association with a mutated genetic profile is not observed. of developing epilepsy (Engel and Pitkänen, 2020). These include a considerable number of neurodegenerative Nucleic acid-based disease biomarkers have represented diseases such as, in the interest of this review, different forms one of the most exciting developments in recent years, with of epilepsy. non-coding RNAs, specifically microRNAs, gaining center stage (Henshall et al., 2016). In this review, we consider the potential DECONVOLUTING CELL-OF-ORIGIN of the still largely unexplored circulating cell-free (cf) DNA. We THROUGH DNA METHYLATION will focus on cfDNA methylation and its ability to predict cell and tissue of origin in epilepsies with a histopathological phenotype The strong association between cfDNA and apoptosis suggests of neuronal damage and death. the occurrence of cell death somewhere in the organism. However, genetic screening of the circulating DNA is unable BIOGENESIS AND THE APOPTOTIC to specifically identify tissue or cell of origin. In this context, CASCADE epigenetics and, specifically, DNA methylation have entered and significantly upgraded cfDNA research. DNA methylation The first description of cfDNA in human plasma dates to 1948 consists of the covalent addition of a methyl group to cytosine (Mandel and Metais, 1948). Nowadays, it is broadly described residues in the genomic sequence. It occurs predominantly as highly degraded DNA fragments circulating freely in the in CpG dinucleotides and is associated with transcriptional peripheral blood. However, the full spectrum of processes regulation. Certain patterns of DNA methylation are highly responsible for cfDNA production and release remains elusive. tissue and cell-specific and conserved in both physiologic and There is a perception that cfDNA may be originated from pathologic conditions (Dor and Cedar, 2018; Figure 1). countless biological features, both physiological and pathological, Alterations of DNA methylation in cfDNA may result from such as inflammation, aging, exercise, or cancer. The main alterations in specific cells or tissue between the pathologic mechanisms of cfDNA release are considered to be active and disease-free settings. Additionally, it can also originate release or cellular breakdown (Aucamp et al., 2018). The overall from changes in the composition of cfDNA due to differential conception, on which most of the studies in this field base cell death rates (Figure 1). The analysis of the cfDNA their hypotheses, is that cfDNA originates predominantly from methylome has been conducted in two main lines of thought. apoptosis. This relies on the matching sizes between cfDNA The first opts for directly calculating differential methylation and DNA fragments resultant from the apoptotic cascade. patterns, such as differentially methylated positions (DMPs) Caspase-activated DNase is one of the main effectors of and differentially methylated regions (DMRs) between different DNA fragmentation during apoptosis. It is a double-stranded conditions (Hatt et al., 2015; Jensen et al., 2015; Legendre endonuclease which lacks exonuclease activity, thus being only et al., 2015; Xu et al., 2017; Gallardo-Gómez et al., 2018; capable of fragmenting DNA in inter-nucleosomal linker regions Gordevičius et al., 2018). However, the direct usage of cfDNA (Heitzer et al., 2020). Human circulating cfDNA presents a profiles as predictors may be associated with an increased predominant and consistent fragment length of approximately signal-to-noise ratio, since DMPs and even DMRs can be 167 bp (Snyder et al., 2016), which corresponds to the length non-specifical and more difficult to interpret biologically. Cell of DNA wrapped around a nucleosome (∼147 bp) plus linker or tissue-of-origin estimation may be considered as a reduction fragments (Heitzer et al., 2020). step, decreasing background noise, and increasing prediction accuracy (Feng et al., 2019). Prediction of cfDNA origin can be GENETIC SEQUENCING AND LIQUID approached through two different strategies. The first consists BIOPSIES of targeted DNA methylation analysis, that is predicting cell- of-origin by searching for previously identified specific DNA In practice, the study of cfDNA has consisted predominantly of methylation alterations characteristic of a cell type (DMPs its genetic characterization. Sequencing of cfDNA is nowadays and DMRs). This approach has been applied throughout a routinely used in some clinic settings. Screening of fetal wide spectrum of settings, including the detection of cfDNA DNA circulating in the mother’s bloodstream, non-invasive derived from pancreatic β-cell in Type 1 diabetic patients Frontiers in Cellular Neuroscience | www.frontiersin.org 2 March 2022 | Volume 16 | Article 852151 Martins-Ferreira et al. Cell-Free DNA Methylation in Epilepsy FIGURE 1 | Cell damage and apoptotic events during both physiological and pathological conditions are thought to release cell-free DNA (cfDNA) into the bloodstream. The pool of cfDNA in circulation present specific DNA methylation patterns that would allow the detection and localization of cell death, opening the doors for a wide spectrum of prospective clinical applications. The selected cell and tissue types are intended to be representative. Hematopoietic cells, the main contributors to cfDNA release, are depicted by monocytes, neutrophils, and lymphocytes. Hepatocytes, pancreatic islet cells, cardiomyocytes, breast, and colon are some examples of tissues with previous evidence on the estimation of cell/tissue of origin based on cfDNA methylation. Brain, neurons, and glia represent the potential of applying such pipelines in neurodegenerative diseases. (Olsen et al., 2016a), cardiomyocytes in cardiac infarction and in the prediction of tissue of origin in cancer patients with no sepsis patients (Zemmour et al., 2018) and hepatocytes in liver clear primary (Cancer of Unknown Primary; Moss et al., 2018). transplant recipients, individuals who underwent hepatectomy An additional interesting aspect of this study is the fact that this and sepsis patients (Lehmann-Werman et al., 2018). The second deconvolution algorithm accounts for primary cortical neurons, approach consists of genome-wide deconvolution tools that have thus allowing its usage in detecting neuronal cell death across been developed to simultaneously estimate the percentages of neurodegenerative diseases. the contribution of multiple tissue and cell types to circulating cfDNA. Deconvolution algorithms have been predominantly BRAIN-DERIVED cfDNA IN used in cancer DNA methylation research (Sun et al., 2015; NEURODEGENERATION Guo et al., 2017; Kang et al., 2017; Li et al., 2018). A study by Moss et al. (2018) developed a comprehensive reference- The application of cfDNA methylation-based analysis to based deconvolution tool accounting for a total of 25 cell/tissue neurodegenerative pathologies are still in their infancy. Research types obtained from 450k and EPIC arrays of primary tissue has consisted mostly of target-based studies, meaning that sources. They validated the previous conception that cfDNA in the detection of circulating brain-derived DNA relies on plasma of healthy individuals as originating predominantly from precisely measuring small regions, with specifically differential hematopoietic cells. Furthermore, the authors demonstrated methylation behavior in the CNS in comparison to other tissues, promising results in pathological contexts, by detecting increased mainly hematopoietic cells. A summary of the current data plasma cfDNA of pancreatic origin in islet transplant recipients, on CNS-derived cfDNA in a neurodegenerative context is significant correlation of hepatocyte-derived cfDNA and clinical presented in Table 1. The first such study that we are aware liver damage markers, and detection of tissue-specific cfDNA of is one by Lehmann-Werman et al. (2016), in which the in three cancer settings (colon, lung, and breast). Interestingly, authors first identified a pattern of clear demethylation in cfDNA estimated origin showed good prognostic ability in two clusters of CpGs in sorted oligodendrocytes’ methylome prostate cancer patients after treatment, and also showed promise (located at the MBP3 gene and in an unannotated region). By Frontiers in Cellular Neuroscience | www.frontiersin.org 3 March 2022 | Volume 16 | Article 852151 Martins-Ferreira et al. Cell-Free DNA Methylation in Epilepsy TABLE 1 | Evidence of CNS-derived cfDNA in neurodegenerative settings. Condition Cell or tissue of DNA methylation Approach Methodology Reference origin pattern MS Oligodendrocyte Demethylation at MBP3 Target-specific Bisulfite sequencing Lehmann-Werman et al. (2016) and unannotated region (CG10809560, Illumina annotation) Cardiac arrest Brain Demethylation at Target-specific Bisulfite sequencing Lehmann-Werman et al. (2016) with brain unannotated region damage (CG09787504, Illumina annotation) TBI Brain Demethylation at Target-specific Bisulfite sequencing Lehmann-Werman et al. (2016) unannotated region (CG09787504, Illumina annotation) MS Oligodendrocyte Demethylation at MOG Target-specific Methylation-specific PCR Olsen et al. (2016b) ALS Brain Increased methylation Target-specific Bisulfite sequencing Mendioroz et al. (2018) at RHBDF promoter AD Brain Increased methylation Target-specific Bisulfite sequencing Pai et al. (2019) at LHX2 Explosive course Neuron and glia Differential DNA Multi-targeted Bisulfite amplicon Chatterton et al. (2021) trainees methylation in sequencing 33 regions AD, Alzheimer’s disease; ALS, Amyotrophic lateral sclerosis; MS, Multiple Sclerosis; TBI, Traumatic Brain Injury. bisulfite sequencing such regions, they described increased multiple profiles of neurotrauma and damage. One can infer oligodendrocyte-derived cfDNA in serum or plasma of that CNS-derived cfDNA shed into the bloodstream may become multiple sclerosis patients. Using the same strategy (known a more accessible and interpretable tool for neurodegenerative unmethylation of a cluster of nine CpGs of brain tissue), biomarker research. they observed an increase in brain-derived cfDNA in cardiac arrest patients with documented brain damage, and also in POTENTIAL OF cfDNA METHYLOME traumatic brain injury patients (Lehmann-Werman et al., 2016). PROFILING IN EPILEPSY In line with this, Olsen et al. (2016b) demonstrated increased demethylation of the MOG gene in sera of active multiple To the best of our knowledge, to date, there are only two sclerosis patients. In amyotrophic lateral sclerosis patients, studies on cfDNA studies in epilepsy. Both analyzing the plasma cfDNA methylation levels of the RHBDF promoter were concentration of cfDNA. Liimatainen et al. (2013) observed increased, relative to healthy controls (Mendioroz et al., 2018). increased sera cfDNA concentration in 167 focal epilepsy Lastly, Alzheimer’s patients have been shown to have higher patients vs. 250 healthy controls. Alapirtti et al. (2016) on the levels of LHX2 methylation in plasma-extracted cfDNA (Pai other hand, failed to verify differences between 52 refractory et al., 2019). epilepsy patients (both focal and generalized) and 250 controls. In a more technically evolved and encompassing study, One of the limitations of these studies is the heterogeneity Chatterton et al. (2021) achieved an interesting proof-of-concept of the studied populations, which include a large spectrum of in which they were able to detect neuron and glia-derived epileptic syndromes. Given the variability within epilepsies and cfDNA in human plasma samples. They developed a bisulfite the increasingly evident differing etiopathogenic fingerprints amplicon sequencing protocol of a total of 33 regions with between syndromes, more narrowly specified epilepsy cohorts neuron and glial-specific DNA methylation patterns identified would be of more relevance. with an in-house pipeline from 450k microarray profiles. In As for cfDNA methylation, nothing has, to the best of blood plasma samples of entry personnel during a training our knowledge, been reported yet. Nonetheless, altered DNA course using explosives, the authors report evidence of neuron methylation patterns in brain tissue of epilepsy patients have and glial cfDNA. Furthermore, significantly increased levels been consistently described (Miller-Delaney et al., 2012; Liu et al., of neuron-derived cfDNA were observed on the day of 2016; Wang et al., 2016; Zhang et al., 2021; Martins-Ferreira training in which exposure to higher pressures was experienced et al., 2022). Major efforts have been made for the use of DNA (Chatterton et al., 2021). methylation as peripheral biomarkers, namely by demonstrating Tissue biopsies are extremely invasive and, in the case of the correlation of DNA methylation patterns in brain tissue neurologic conditions, particularly inaccessible. Cerebrospinal and peripheral tissues, such as blood, saliva, and buccal mucosa fluid (CSF) analysis is an option but still causes major discomfort (Braun et al., 2019). Reinforcing the aforementioned idea, cfDNA to patients. Along the neurodegeneration spectrum, there are methylation may be more representative of a specific pathological Frontiers in Cellular Neuroscience | www.frontiersin.org 4 March 2022 | Volume 16 | Article 852151 Martins-Ferreira et al. Cell-Free DNA Methylation in Epilepsy FIGURE 2 | Epileptogenesis is considered to follow a progressive model. After the initial precipitating injury (IPI), the brain parenchyma enters a latent phase in which the ability to generate spontaneous recurrent seizures (SRSs) is established. In the chronic stage, upon initiation of unprovoked seizure activity, epileptogenic molecular and structural remodeling progress with time. DNA methylation profiling along the different timeframes of epileptogenesis would potentially represent a valuable approach for the development of novel non-invasive biomarkers. Possible analytical strategies include detection of altered cfDNA methylation patterns in pathological settings in comparison to controls, or estimation of the percentage of tissue or cell type contribution to the circulating cfDNA pool, which potentially insights on occurring CNS-specific cell death in the early stages of the disease. MSP, Methylation-Specific PCR; RRBS, reduced representation bisulfite sequencing; WBGS, whole genome bisulfite sequencing. Frontiers in Cellular Neuroscience | www.frontiersin.org 5 March 2022 | Volume 16 | Article 852151 Martins-Ferreira et al. Cell-Free DNA Methylation in Epilepsy phenotype due to the link between cfDNA methylome, differing monitoring, and provide for therapeutic outcome prediction of tissue/cell contribution, and pattern of cell death, as described (Figure 2). throughout this review. Mesial Temporal Lobe Epilepsy (MTLE) is one of the most CONCLUDING REMARKS studied epilepsies due to its high incidence (it is the most common focal epilepsy in adults) and its high refractoriness rates. The research on circulating cfDNA has evolved exponentially Hippocampal sclerosis is the most prevalent histopathological during the last decades, from the proven clinical utility of feature in MTLE. It is characterized by an exacerbated state genetic characterization to the emerging applications of DNA of gliosis and a severe neuronal death landscape in the mesial methylation. Identification of biomarkers is an urgent need temporal regions (Blümcke et al., 2013). A pertinent question across various pathologies, particularly in neurologic diseases in regarding MTLE’s etiopathology is the time frame of HS which tissue biopsies are extremely difficult to obtain or simply occurrence. It has been considered a consequence of recurrent inaccessible. We propose that cfDNA methylation should be exposure to seizure activity. However, it can also be speculated investigated in epileptic patients, as we consider it to be and that HS may have a role in promoting pro-epileptogenic intriguing and promising beam of light in what has been the activity onset and progression. This is, in part, due to the gloomy field of epilepsy biomarker research. fact that the diagnosis often occurs in the chronic stage, upon the appearance of spontaneous recurrent seizures. The most recently reviewed definition of epileptogenesis describes AUTHOR CONTRIBUTIONS it as a continuous event. After the initial precipitating injury, All authors were responsible for the development of the the brain undergoes a latent phase in which the ability to conceptual map. RM-F performed literature search and drafted produce spontaneous recurrent seizures is acquired. The chronic the manuscript. BL and PC were responsible for the critical stage, initiated upon the first unprovoked seizure, is no longer revision and final approval of the final version to be published. considered stationary, as was previously the case. It is rather All authors contributed to the article and approved the submitted a progressive phenomenon, with pathological molecular and version. cellular alterations extending as the disease progresses (Pitkänen et al., 2016). A recent study by our group has demonstrated major DNA methylation alterations in both hippocampus and anterior FUNDING temporal neocortex of MTLE-HS patients in comparison to autopsied non-epileptic controls, with enrichment for a wide RM-F was funded by an FCT (Fundação para a Ciência e spectrum of epileptogenesis-related pathways. Additionally, Tecnologia) fellowship (grant number SFRH/BD/137900/2018). we demonstrate a potential progressive remodeling of DNA Unit for Multidisciplinary Research in Biomedicine (UMIB) was methylation in inflammatory genes with increased disease funded by FCT Portugal (grant numbers UIDB/00215/2020 duration (Martins-Ferreira et al., 2022). and UIDP/00215/2020), and Laboratory for Integrative The main argument of interest of this review focus on and Translational Research in Population Health (ITR) the fact that cell death occurs in the MTLE-HS brain. (LA/P/0064/2020). Despite the time-point at which it may be occurring. Thus, it is plausible that neuron- or glia-derived cfDNA may be ACKNOWLEDGMENTS released into the circulation during neuro damage. We call for novel cfDNA methylation screening studies in epileptic We acknowledge Professor Berta Martins da Silva and the patients, from targeted approaches like bisulfite sequencing remaining members of the Immunogenetics Laboratory and methylation-specific PCR to genome-wide screenings of the Molecular Pathology and Immunology Department using microarrays, whole-genome bisulfite sequencing, of ICBAS-UP. We thank the Neurology, Neurophysiology and reduced representation bisulfite sequencing. Such and Neurosurgery Departments of Hospital de Santo studies would be potentially helpful in all stages of the António—Centro Hospitalar e Universitário do Porto. We epileptogenic landscape. It could promote detection of patients also acknowledge Professor Esteban Ballestar and all members of at high risk of epileptogenesis after a brain trauma incident Epigenetics and Immune Disease of the Josep Carreras Research and during latency, facilitate earlier diagnosis and better Institute (IJC). REFERENCES the human body: a critical re-evaluation of the literature. Biol. Rev. Camb. Philos. Soc. 93, 1649–1683. doi: 10.1111/brv. Alapirtti, T., Jylhävä, J., Raitanen, J., Mäkinen, R., Peltola, J., Hurme, M. A., et al. 12413 (2016). The concentration of cell-free DNA in video-EEG patients is dependent Blümcke, I., Thom, M., Aronica, E., Armstrong, D. D., Bartolomei, F., on the epilepsy syndrome and duration of epilepsy. Neurol. Res. 38, 45–50. Bernasconi, A., et al. (2013). International consensus classification of doi: 10.1080/01616412.2015.1127004 hippocampal sclerosis in temporal lobe epilepsy: a task force report from Aucamp, J., Bronkhorst, A. J., Badenhorst, C. P. S., and Pretorius, P. J. the ILAE commission on diagnostic methods. Epilepsia 54, 1315–1329. (2018). The diverse origins of circulating cell-free DNA in doi: 10.1111/epi.12220 Frontiers in Cellular Neuroscience | www.frontiersin.org 6 March 2022 | Volume 16 | Article 852151 Martins-Ferreira et al. Cell-Free DNA Methylation in Epilepsy Braun, P. R., Han, S., Hing, B., Nagahama, Y., Gaul, L. N., Heinzman, J. T., using methylation patterns of circulating DNA. Proc. Natl. Acad. Sci. U S A 113, et al. (2019). Genome-wide DNA methylation comparison between live E1826–1834. doi: 10.1073/pnas.1519286113 human brain and peripheral tissues within individuals. Transl. Psychiatry 9:47. Li, W., Li, Q., Kang, S., Same, M., Zhou, Y., Sun, C., et al. (2018). Cancerdetector: doi: 10.1038/s41398-019-0376-y ultrasensitive and non-invasive cancer detection at the resolution of individual Chabon, J. J., Hamilton, E. G., Kurtz, D. M., Esfahani, M. S., Moding, E. J., reads using cell-free DNA methylation sequencing data. Nucleic Acids Res. Stehr, H., et al. (2020). Integrating genomic features for non-invasive 46:e89. doi: 10.1093/nar/gky423 early lung cancer detection. Nature 580, 245–251. doi: 10.1038/s41586-020- Liimatainen, S. P., Jylhävä, J., Raitanen, J., Peltola, J. T., and Hurme, M. A. 2140-0 (2013). The concentration of cell-free DNA in focal epilepsy. Epilepsy Res. 105, Chatterton, Z., Mendelev, N., Chen, S., Carr, W., Kamimori, G. H., Ge, Y., et al. 292–298. doi: 10.1016/j.eplepsyres.2013.03.005 (2021). Bisulfite amplicon sequencing can detect glia and neuron cell-free DNA Liu, X., Ou, S., Xu, T., Liu, S., Yuan, J., Huang, H., et al. (2016). New in blood plasma. Front. Mol. Neurosci. 14:672614. doi: 10.3389/fnmol.2021. differentially expressed genes and differential DNA methylation underlying 672614 refractory epilepsy. Oncotarget 7, 87402–87416. doi: 10.18632/oncotarget. Dor, Y., and Cedar, H. (2018). Principles of DNA methylation and 13642 their implications for biology and medicine. Lancet 392, 777–786. Mandel, P., and Metais, P. (1948). [Nuclear acids in human blood plasma]. doi: 10.1016/S0140-6736(18)31268-6 Comptes rendus des seances de la Societe de biologie et de ses filiales 142, Engel, J, Jr., and Pitkänen, A. (2020). Biomarkers for epileptogenesis and its 241–243. treatment. Neuropharmacology 167:107735. doi: 10.1016/j.neuropharm.2019. Martins-Ferreira, R., Leal, B., Chaves, J., Li, T., Ciudad, L., Rangel, R., et al. (2022). 107735 Epilepsy progression is associated with cumulative DNA methylation changes Fan, H. C., Gu, W., Wang, J., Blumenfeld, Y. J., El-Sayed, Y. Y., and Quake, S. R. in inflammatory genes. Prog. Neurobiol. 209:102207. doi: 10.1016/j.pneurobio. (2012). Non-invasive prenatal measurement of the fetal genome. Nature 487, 2021.102207 320–324. doi: 10.1038/nature11251 Mendioroz, M., Martínez-Merino, L., Blanco-Luquin, I., Urdánoz, A., Roldán, M., Feng, H., Jin, P., and Wu, H. (2019). Disease prediction by cell-free and Jericó, I. (2018). Liquid biopsy: a new source of candidate biomarkers DNA methylation. Brief Bioinform. 20, 585–597. doi: 10.1093/bib/ in amyotrophic lateral sclerosis. Ann. Clin. Transl. Neurol. 5, 763–768. bby029 doi: 10.1002/acn3.565 Gallardo-Gómez, M., Moran, S., de la Cadena, M. P., Martínez-Zorzano, V. S., Miller-Delaney, S. F. C., Das, S., Sano, T., Jimenez-Mateos, E. M., Bryan, K., Rodríguez-Berrocal, F. J., Rodríguez-Girondo, M., et al. (2018). A new Buckley, P. G., et al. (2012). Differential DNA methylation patterns approach to epigenome-wide discovery of non-invasive methylation define status epilepticus and epileptic tolerance. J. Neurosci. 32, 1577–1588. biomarkers for colorectal cancer screening in circulating cell-free DNA doi: 10.1523/JNEUROSCI.5180-11.2012 using pooled samples. Clin. Epigenetics 10:53. doi: 10.1186/s13148-018- Moss, J., Magenheim, J., Neiman, D., Zemmour, H., Loyfer, N., Korach, A., 0487-y et al. (2018). Comprehensive human cell-type methylation atlas reveals origins Gordevičius, J., Kriščiūnas, A., Groot, D. E., Yip, S. M., Susic, M., Kwan, A., et al. of circulating cell-free DNA in health and disease. Nat. Commun. 9:5068. (2018). Cell-free DNA modification dynamics in abiraterone acetate-treated doi: 10.1038/s41467-018-07466-6 prostate cancer patients. Clin. Cancer Res. 24, 3317–3324. doi: 10.1158/1078- Nabet, B. Y., Esfahani, M. S., Moding, E. J., Hamilton, E. G., Chabon, J. J., 0432.CCR-18-0101 Rizvi, H., et al. (2020). Noninvasive early identification of therapeutic benefit Guo, S., Diep, D., Plongthongkum, N., Fung, H.-L., Zhang, K., and Zhang, K. from immune checkpoint inhibition. Cell 183, 363–376.e13. doi: 10.1016/j.cell. (2017). Identification of methylation haplotype blocks aids in deconvolution of 2020.09.001 heterogeneous tissue samples and tumor tissue-of-origin mapping from plasma Olsen, J. A., Kenna, L. A., Spelios, M. G., Hessner, M. J., and Akirav, E. M. (2016a). DNA. Nat. Genet. 49, 635–642. doi: 10.1038/ng.3805 Circulating differentially methylated amylin DNA as a biomarker of β-cell Hatt, L., Aagaard, M. M., Graakjaer, J., Bach, C., Sommer, S., Agerholm, I. E., et al. loss in type 1 diabetes. PLoS One 11:e0152662. doi: 10.1371/journal.pone.01 (2015). Microarray-based analysis of methylation status of CpGs in placental 52662 DNA and maternal blood DNA – potential new epigenetic biomarkers for cell Olsen, J. A., Kenna, L. A., Tipon, R. C., Spelios, M. G., Stecker, M. M., and free fetal DNA-based diagnosis. PLoS One 10:e0128918. doi: 10.1371/journal. Akirav, E. M. (2016b). A minimally-invasive blood-derived biomarker of pone.0128918 oligodendrocyte cell-loss in multiple sclerosis. EBioMedicine 10, 227–235. Heitzer, E., Auinger, L., and Speicher, M. R. (2020). Cell-free DNA and apoptosis: doi: 10.1016/j.ebiom.2016.06.031 how dead cells inform about the living. Trends Mol. Med. 26, 519–528. Pai, M.-C., Kuo, Y.-M., Wang, I.-F., Chiang, P.-M., and Tsai, K.-J. (2019). doi: 10.1016/j.molmed.2020.01.012 The role of methylated circulating nucleic acids as a potential biomarker in Henshall, D. C., Hamer, H. M., Pasterkamp, R. J., Goldstein, D. B., Kjems, J., Alzheimer’s disease. Mol. Neurobiol. 56, 2440–2449. doi: 10.1007/s12035-018 Prehn, J. H. M., et al. (2016). MicroRNAs in epilepsy: pathophysiology -1229-z and clinical utility. Lancet Neurol. 15, 1368–1376. doi: 10.1016/S1474- Pitkänen, A., Löscher, W., Vezzani, A., Becker, A. J., Simonato, M., Lukasiuk, K., 4422(16)30246-0 et al. (2016). Advances in the development of biomarkers for epilepsy. Lancet Jensen, T. J., Kim, S. K., Zhu, Z., Chin, C., Gebhard, C., Lu, T., et al. Neurol. 15, 843–856. doi: 10.1016/S1474-4422(16)00112-5 (2015). Whole genome bisulfite sequencing of cell-free DNA and its cellular Pitkänen, A., Lukasiuk, K., Dudek, F. E., and Staley, K. J. (2015). Epileptogenesis. contributors uncovers placenta hypomethylated domains. Genome Biol. 16:78. Cold Spring Harb. Perspect. Med. 5:a022822. doi: 10.1101/cshperspect.a0 doi: 10.1186/s13059-015-0645-x 22822 Kang, S., Li, Q., Chen, Q., Zhou, Y., Park, S., Lee, G., et al. (2017). Cancerlocator: Powles, T., Assaf, Z. J., Davarpanah, N., Banchereau, R., Szabados, B. E., non-invasive cancer diagnosis and tissue-of-origin prediction using Yuen, K. C., et al. (2021). CtDNA guiding adjuvant immunotherapy methylation profiles of cell-free DNA. Genome Biol. 18:53. doi: 10.1186/s13059- in urothelial carcinoma. Nature 595, 432–437. doi: 10.1038/s41586-021- 017-1191-5 03642-9 Legendre, C., Gooden, G. C., Johnson, K., Martinez, R. A., Liang, W. S., Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M., and Shendure, J. (2016). Cell- and Salhia, B. (2015). Whole-genome bisulfite sequencing of cell-free DNA free DNA comprises an in vivo nucleosome footprint that informs its tissues- identifies signature associated with metastatic breast cancer. Clin. Epigenetics of-origin. Cell 164, 57–68. doi: 10.1016/j.cell.2015.11.050 7:100. doi: 10.1186/s13148-015-0135-8 Sun, K., Jiang, P., Allen Chan, K. C., Wong, J., Cheng, Y. K. Y., Liang, R. H. S., Lehmann-Werman, R., Magenheim, J., Moss, J., Neiman, D., Abraham, O., et al. (2015). Plasma DNA tissue mapping by genome-wide methylation Piyanzin, S., et al. (2018). Monitoring liver damage using hepatocyte-specific sequencing for noninvasive prenatal, cancer and transplantation assessments. methylation markers in cell-free circulating DNA. JCI Insight 3:e120687. Proc. Natl. Acad. Sci. U S A 112, E5503–5512. doi: 10.1073/pnas.15087 doi: 10.1172/jci.insight.120687 36112 Lehmann-Werman, R., Neiman, D., Zemmour, H., Moss, J., Magenheim, J., De Vlaminck, I., Valantine, H. A., Snyder, T. M., Strehl, C., Cohen, G., Luikart, H., Vaknin-Dembinsky, A., et al. (2016). Identification of tissue-specific cell death et al. (2014). Circulating cell-free DNA enables noninvasive diagnosis of heart Frontiers in Cellular Neuroscience | www.frontiersin.org 7 March 2022 | Volume 16 | Article 852151 Martins-Ferreira et al. Cell-Free DNA Methylation in Epilepsy transplant rejection. Sci. Transl. Med. 6:241ra77. doi: 10.1126/scitranslmed. Conflict of Interest: The authors declare that the research was conducted in the 3007803 absence of any commercial or financial relationships that could be construed as a Wang, L., Fu, X., Peng, X., Xiao, Z., Li, Z., Chen, G., et al. (2016). DNA potential conflict of interest. methylation profiling revealss correlation of differential methylation patterns with gene expression in human epilepsy. J. Mol. Neuroscience. 59, 68–77. Publisher’s Note: All claims expressed in this article are solely those of the authors doi: 10.1007/s12031-016-0735-6 and do not necessarily represent those of their affiliated organizations, or those of Xu, R.-H., Wei, W., Krawczyk, M., Wang, W., Luo, H., Flagg, K., et al. (2017). the publisher, the editors and the reviewers. Any product that may be evaluated in Circulating tumour DNA methylation markers for diagnosis and prognosis of this article, or claim that may be made by its manufacturer, is not guaranteed or hepatocellular carcinoma. Nat. Mater. 16, 1155–1161. doi: 10.1038/nmat4997 endorsed by the publisher. Zemmour, H., Planer, D., Magenheim, J., Moss, J., Neiman, D., Gilon, D., et al. (2018). Non-invasive detection of human cardiomyocyte death Copyright © 2022 Martins-Ferreira, Leal and Costa. This is an open-access article using methylation patterns of circulating DNA. Nat. Commun. 9:1443. distributed under the terms of the Creative Commons Attribution License (CC BY). doi: 10.1038/s41467-018-03961-y The use, distribution or reproduction in other forums is permitted, provided the Zhang, W., Wang, H., Liu, B., Jiang, M., Gu, Y., Yan, S., et al. (2021). original author(s) and the copyright owner(s) are credited and that the original Differential DNA methylation profiles in patients with temporal lobe epilepsy publication in this journal is cited, in accordance with accepted academic practice. and hippocampal sclerosis ILAE type I. J. Mol. Neurosci. 71, 1951–1966. No use, distribution or reproduction is permitted which does not comply with doi: 10.1007/s12031-020-01780-9 these terms. Frontiers in Cellular Neuroscience | www.frontiersin.org 8 March 2022 | Volume 16 | Article 852151