Differential DNA Methylation Patterns in Patients with Epilepsy due to Malformations of Cortical Development: A Pilot Study
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.273638
Source of Support: None, Conflict of Interest: None
Keywords: DNA methylation, malformations of cortical development, neurogenetics, refractory epilepsy
Malformations of cortical development (MCDs) are a leading cause of medically refractory focal epilepsy and can be found in as high as 40% of these patients. MCDs can be linked with mutations affecting various different stages of cortical development. Additionally, somatic mutations have been found in patients with certain types of MCDs, including double cortex syndrome, hemimegalencephaly, and pachygyria in the absence of a germline mutation. However, somatic and germline mutations can be identified in only a limited number of patients. Moreover, the clinical presentation of MCDs is quite heterogeneous in terms of age of presentation, drug responsiveness, and impact on cognitive development.
Epigenetic alteration encompasses heritable changes in gene expression that does not involve modifications to the underlying DNA sequence – a change in phenotype without a change in genotype. DNA methylation is one of three mediators of epigenetics, the other two being noncoding RNAs and histone acetylation. A switch between de novo DNA methylation and demethylation during development results in a stable and heritable change in gene expression patterns. Animal knockout models have shown the control of methylation and demethylation on the development of the brain. Data from experimental studies as well as human temporal lobe epilepsy postulate that epigenetic regulation of gene expression has a strong contribution in epileptogenesis. This study was aimed at comparative analysis of DNA methylation profile in brain tissue of children with refractory focal epilepsy who underwent resective epilepsy surgery, comparing those with MCD to children who did not have radiological or histopathological evidence of an MCD (non-MCD).
We selected postsurgical brain tissue samples of 13 children with refractory focal epilepsy, who underwent resective epilepsy surgery at Children's Hospital of Michigan. Study was approved by institutional review board and informed consent was obtained from each patient families. The candidacy for surgery was based on medical refractoriness, electrophysiological monitoring, anatomic and functional neuroimaging using magnetic resonance imaging, and 18-flourodeoxyglucose positron emission tomography (18-FDG PET) scan as well as electrocorticographic data using subdural grid and strip electrodes with capturing of habitual seizures. Clinical, neuroimaging and pathological data about subjects were collected by retrospective review of electronic medical record. Methylation analysis was performed on the cortical tissue using Illumina® 450k Methylation Microarray. Data analysis was performed using an in-house workflow developed using open-source R statistical packages. The data were preprocessed by log2 transformation and quantile normalization. Pathway analysis was performed using the R/Bioconductor package “generally applicable gene-set/pathway analysis” (gage). The list of top driver genes was obtained from Catalogue of Somatic Mutations in Cancer database. This gene list was intersected with genes differentially expressed between MCD and non-MCD tissues to identify the driver genes that could be potentially associated.
Clinical characterisation of patients is summarized in [Table 1].
Thirty-one genes were found to be hypermethylated in the MCD group when compared with the non-MCD group. Using gage analysis, the top genes that were differentially methylated in patients with MCD included genes linked to Ephrin–Reelin pathway, potassium channels, and glutathione metabolism [Table 2].
Our preliminary study with a relatively small sample size suggests that DNA methylation of some potential genes might be involved in the pathobiogenesis of MCDs in patients with refractory epilepsy. We identified genes with differential methylation in pathways that play a vital role in the structural organization as well as the function of the cerebral cortex as discussed below.
Reelin–Ephrin pathway (EFNB3 and EPHB1)
Reelin regulates migration of neurons during brain development. Derangements of this pathway have been associated with lissencephaly. A previous study also noted that the level of Ephrin B receptor protein and mRNA is upregulated as compared with controls in rats as well as patients with temporal lobe epilepsy. Our study provides further data suggesting the role of Ephrin B receptor in cortical malformations by indicating that this change may be epigenetic in nature in some patients and can be heritable.
Genes linked to potassium channels (KCNQ5 and KCNG4)
The role of potassium channels in maintaining neuronal excitability and its association with neurological disorders is well described. However, the two potassium channel genes that appeared in our cohort have not been extensively associated with epilepsy. A single report of four probands with intellectual disability and refractory epilepsy with missense mutations in KCNQ5 gene has been described. This gene codes for a potassium channel that is widely expressed in the brain and generates M-type currents. An exhaustive literature search regarding KCNG4 gene showed that it has never been incriminated as a culprit in seizure disorders; but rather, variants in this gene were discovered in migraine probands. Although none of our patients had a history of migraine, the clinical impact of epigenetic regulation of this gene on MCDs and epileptogenicity needs to be further explored.
Genes linked to glutathione metabolism (GSTM5, GSTM1, and GSTO2)
Glutathione is a potent antioxidant vital in maintaining intracellular equilibrium of reactive oxygen species. Its role in aging, neurodegenerative diseases, and cancer has been studied extensively. A study done on 26 patients with progressive myoclonus epilepsy has shown that polymorphisms in GST genes are predictive of the phenotype in this condition. Given the methylation changes in genes involved in glutathione metabolism at the tissue level as seen in our patients, we hypothesize that this pathway may be crucial in epileptogenesis in children with MCDs.
A major limitation of our study is that our patients' blood samples were not tested for germline mutations. In addition, histopathology and neuroimaging could have theoretically missed subtle changes associated with MCDs in the second group, which formed our control. Also, given the heterogeneity of our results and the small sample size, we were unable to correlate the impact of these epigenetic signatures on other clinical characteristics of these patients including developmental profile, age of presentation, etc., The clinical implications of this study are uncertain at this time, but a few optimistic predictions can be made if the results are validated by larger studies:
Unique patterns of methylation are observed in children with refractory focal epilepsy with MCD compared with those who do not have MCD. In addition to changes in cell migration pathways (Ephrin), reduced expression of potassium channel-related genes in patients with MCD may cause increased cortical excitability and account for epileptogenicity. Tissue-specific oxidative injury as seen with GST methylation may be a cause or effect of seizures in these patients. Further studies with a larger sample size may help elucidate the pathophysiological and clinical significance of these patterns.
Financial support and sponsorship
Children's Hospital of Michigan Foundation; Grant number- R1-2016-63.
Conflicts of interest
There are no conflicts of interest.
[Table 1], [Table 2]