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

Objective: 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. Methods: 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; > 90x) 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 (10x). Results 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 similar to 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. Conclusions: 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 similar to 80% for the entire FCDII cohort.

Automated summary

In this study, we aimed to investigate the genetic etiology of focal cortical dysplasia type II (FCDII) by detecting brain somatic mutations in mTOR pathway genes. Despite the use of deep sequencing and analytics, about 40% of cases remained genetically unexplained. By isolating cells exhibiting phosphorylation of the S6 ribosomal protein (p-S6) in brain tissues and performing whole-genome sequencing, we found ultra-low level somatic mutations and germline structural variations in a significant portion of mutation-negative FCDII patients. Our method significantly increased the genetic diagnostic rate for FCDII.