Abstracts

Rationale: Epilepsy can be caused by somatic mutations occurring post-zygotically, and a subset of these mutations are restricted to brain. Brain-only somatic mutations are detectable in resected brain tissue available from a minority of epilepsy patients. Cell-free DNA (cfDNA) shed from brain into cerebrospinal fluid (CSF) is an established “liquid biopsy” for brain tumour mutation detection, and may provide an alternative route for detection of somatic mosaicism in epileptic brain. This study aimed to quantify CSF cfDNA levels in epilepsy patients and provide proof of principle that CSF can be used as a surrogate for brain tissue to provide molecular diagnosis for epilepsy. Methods: For cfDNA quantification, CSF was collected from 28 lesional epilepsy patients via neurosurgery and 28 control patients without seizure via lumbar puncture. CSF cfDNA was quantified with droplet digital PCR using a commercial assay to interrogate GNAQ c.548G wildtype allele as a reference for cfDNA level. For molecular diagnosis, CSF was obtained from a patient with subcortical band heterotopia (SBH) and temporal lobe epilepsy (TLE) via lumbar puncture. An in-house droplet digital PCR assay was used to detect the somatic LIS1 p.K64X mutation in this patient’s CSF cfDNA. Results: cfDNA was reliably detected in all CSF samples and PCR-based analysis confirmed all DNA fragments were ≤ 200 bp. There was a 4.19-fold increase in the mean value of cfDNA concentration in epilepsy patients compared to controls (562.01 copies/mL CSF vs. 134.07 copies/mL CSF). Mann-Whitney U test indicated the increase in epilepsy patients was statistically significant (p < 0.0001). In the patient with SBH and TLE, mosaic LIS1 p.K64X mutation was detected in CSF cfDNA with an allele frequency of 9.4%. Conclusions: Our data indicate that there is sufficient cfDNA in CSF from epilepsy patients to make liquid biopsy viable. In one patient we demonstrated proof of principle that CSF liquid biopsy can be used as a surrogate for brain tissue to detect somatic mosaicism. CSF analysis may provide a novel means of diagnosing brain somatic mutations as a cause of epilepsy. Funding: This study was supported by National Health and Medical Research Council Program Grant (1091593) to I.E.S. and S.F.B., a Project Grant (1129054) to S.F.B., a Project Grant (1079058) to M.S.H., a Practitioner Fellowship (1006110) to I.E.S., a R.D Wright Career Development Fellowship (1063799) to M.S.H, and a Program Grant (1054618) and Senior Research Fellowship (1002098) to M.B. R.J.L. is supported by a Melbourne Children’s Clinician Scientist Fellowship. Z.Y. is supported by a University of Melbourne Australian Postgraduate Award International Graduate Research Training Scholarship, and a private scholarship from the Tang Lixin Education Development Fund. M.B. was supported by the Victorian Government’s Operational Infrastructure Support Program and Australian Government NHMRC IRIIS.