The majority of non-acquired focal epilepsies (NAFE) have a presumed genetic etiology. However, the current yield of diagnostic genetic testing in affected patients is very low. This diagnostic gap is an important barrier to the broader use of gene-targeted therapies. Increasing evidence from resected brain tissue of individuals with NAFE points towards an important role of pathogenic somatic variants and methylation abnormalities. Most NAFE patients however do not undergo brain surgery, and the lack of brain tissue precludes a genetic and histopathological diagnosis. In this project, we aim to prove that cell-free DNA (cfDNA) circulating in cerebrospinal fluid (CSF) and serum of patients with NAFE, including individuals with malformations of cortical development (MCD), can be used to bridge this diagnostic gap. First, we will apply a deep- sequencing protocol to detect novel somatic variants in cfDNA from CSF. Second, we will use nanopore sequencing on native cfDNA to both identify somatic copy number changes and to classify patients using aberrant cfDNA methylation patterns consistent with different subtypes of MCD and focal epilepsy. By prioritizing patients who undergo epilepsy surgery for our study cohort, we will validate the detected somatic and epigenetic changes in paired brain tissue, thereby providing an estimate of reliability for our analytical approach. By identifying the genetic etiology and disease methylation profiles from cfDNA, we can improve diagnostic yield and clinical decision-making in this severely affected population.

Promising and Challenging Fundamental Science: One third of epilepsy patients are therapy resistant, and a genetic diagnosis has been shown to enable more targeted treatment choices. However, two thirds of these patients remain without a definitive genetic diagnosis. In this project, we will unmask the hidden genetic causes of NAFE by implementing a first-of-its-kind approach that searches both for somatic single nucleotide or copy number variants, and their effective pathologic changes to DNA methylation patterns. This project pushes scientific and clinical boundaries: it will establish and validate novel sequencing methods that in turn pave the way for better diagnosis, classification, and treatment for the large group of drug-resistant focal epilepsy patients who do not undergo epilepsy surgery.

Cross-Belgian Collaboration: This project brings together the expertise and clinical reach of top neurogenetic and neurological teams across four medical centers in Flanders and Wallonia. In doing so, this project gains access to a unique and well-phenotyped patient cohort that is integral to the success of this project.