Abstracts

This abstract has been invited to present during the Genetics & Behavior/Neuropsychology/Language platform session
This abstract has been invited to present during the Basic Science Poster Highlights poster session

Rationale: Brain somatic mutations (including the two-hit model) in mTOR pathways genes are a major genetic etiology of focal cortical dysplasia (FCD) type II presenting dysmorphic neurons and cortical dyslamination with intractable focal epilepsy. However, despite the development of deep sequencing in patients’ brain tissues and bioinformatic analysis tools detecting low-level somatic mutations, ~40% of FCDII still remains genetically unexplained.

Methods: We optimized the cell isolation method stained with phosphorylation of S6 ribosomal protein (p-S6) from frozen brain tissues using fluorescence-activated cell sorting (FACS). We sequenced deep whole genome sequencing (WGS; >90X read depth) in p-S6⁺ cells in 11 mutation-negative FCDII patients in deep sequencing of bulky brain tissues. We then performed targeted gene sequencing of whole genome amplified samples from FACS-enriched cells and low-read depth WGS (10X) in an independent cohort of 10 mutation-negative FCDII patients.

Results: We found that 28.6% (6 of 21) of mutation-negative FCDII in deep sequencing of bulky brain tissues carries ultra-low level somatic mutations (e.g., less than 0.2% of variant allele frequency; VAF) in mTOR pathway genes. Our method showed ~34 times increase of the average mutational burden in FACS mediated enrichment of p-S6⁺ cells (average VAF: 5.84%) from mutation-negative FCDII, compared to that in bulky brain tissues (average VAF: 0.17%). Also, we found that 19% (4 of 21) of mutation-negative FCDII carried germline structural variations in NPRL3 or DEPDC5 undetectable in the whole exome or targeted gene sequencing.

Conclusions: Overall, our study shows that the detection of ultra-low level somatic mutations and germline structural variations increases the genetic diagnostic rate by up to an overall ~80% of the entire FCD II cohorts. Our method alleviates the limitations of the current diagnosis, and for clinical practice, can be considered as the genetic test for mutation-negative FCDII cases in the conventional deep sequencing of bulky brain tissues.

Funding: Suh Kyungbae Foundation (to JHL), from the National Research Foundation of Korea funded by the Korea government, Ministry of Science and ICT (Grant No. 2019R1A3B2066619 [to JHL]), and the National Research Foundation (NRF-2019H1A2A1077149 [to JHK])