Ultra-Low Level Somatic Mutations and Structural Variations in Focal Cortical Dysplasia Type II

Brain somatic mutations in mTOR pathway genes are a major genetic etiology of focal cortical dysplasia type II (FCDII). Despite a greater ability to detect low-level somatic mutations in the brain by deep sequencing and analytics, about 40% of cases remain genetically unexplained.

We included 2 independent cohorts consisting of 21 patients with mutation-negative FCDII without apparent mutations on conventional deep sequencing of bulk brain. To find ultra-low level somatic variants or structural variants, we isolated cells exhibiting phosphorylation of the S6 ribosomal protein (p-S6) in frozen brain tissues using fluorescence-activated cell sorting (FACS). We then performed deep whole-genome sequencing (WGS; >90×) in p-S6⁺ cells in a cohort of 11 patients with mutation-negative. Then, we simplified the method to whole-genome amplification and target gene sequencing of p-S6⁺ cells in independent cohort of 10 patients with mutation-negative followed by low-read depth WGS (10×).

We found that 28.6% (6 of 21) of mutation-negative FCDII carries ultra-low level somatic mutations (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%) than in bulky brain tissues (average VAF = 0.17%). We found that 19% (4 of 21) carried germline structural variations in GATOR1 complex undetectable in whole exome or targeted gene sequencing.

Our method facilitates the detection of ultra-low level somatic mutations, in specifically p-S6⁺ cells, and germline structural variations and increases the genetic diagnostic rate up to ~80% for the entire FCDII cohort. ANN NEUROL 2023;93:1082-1093.