Abstracts

Rationale: Somatic mosaicism in a fraction of brain cells causes neurodevelopmental disorders, including childhood intractable epilepsy. Although a single cell with harmful somatic mutations is unlikely to cause widespread brain dysfunction such as epilepsy, the exact level of somatic mutation burden that disrupts the brain function has remained unexplored thus far.


Methods: Using in utero electroporation of mTOR mutant plasmids, we induced various mosaic burdens in focal cortical dysplasia type II (FCD II) mice, featuring mTOR somatic mosaicism and spontaneous behavioral seizures. Imaging and three-dimensional reconstruction analysis of cortical slices was performed to examine the mosaic burdens ranging from approximately 1,000 to 40,000 neurons expressing the mTOR mutant in the somatosensory (SSC) or medial prefrontal (PFC) cortex. Video-EEG monitoring was conducted to correlate the severity of seizures and mosaic burdens. Ultra-deep amplicon sequencing was performed to validate the existence of extremely low-level mosaicism in previously mutation-negative FCDII patient’s brain tissues.


Results: Remarkably, approximately 8,000 to 9,000 neurons expressing the MTOR mutant, which are extrapolated to constitute 0.08-0.09% of total cells or roughly 0.04% of variant allele frequency (VAF) in the mouse hemicortex, were sufficient to trigger epileptic seizures. The mutational burden was correlated with seizure frequency and onset, with a higher tendency for electrographic inter-ictal spikes and beta- and gamma-frequency oscillations in FCD II mice exceeding the threshold. Moreover, mutation-negative FCD II patients in deep sequencing of their bulky brain tissues revealed somatic mosaicism of the mTOR pathway genes as low as 0.07% in resected brain tissues through ultra-deep targeted sequencing (up to 20 million reads).


Conclusions: Our study suggests that extremely low levels of somatic mosaicism (such as less than 0.1% VAF) can contribute to brain dysfunction with focal epilepsy.


Funding: This study was supported by grants from the Suh Kyungbae Foundation, and by the National Research Foundation of Korea funded by the Korean government’s Ministry of Science and ICT (Grant No. 2019R1A3B2066619). Also, this work was also supported by a grant from the M.D., Ph.D./Medical Scientist Training Program through the Korea Health Industry Development Institute funded by the Ministry of Health and Welfare of the Republic of Korea.