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

Ann Neurol. 2023 Jun;93(6):1082-1093. doi: 10.1002/ana.26609. Epub 2023 Feb 8.

Abstract

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; >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×).

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 ~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 ~80% for the entire FCDII cohort. ANN NEUROL 2023;93:1082-1093.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Epilepsy* / genetics
  • Focal Cortical Dysplasia*
  • Humans
  • Malformations of Cortical Development, Group I
  • Mutation / genetics
  • TOR Serine-Threonine Kinases / genetics

Substances

  • TOR Serine-Threonine Kinases

Supplementary concepts

  • Focal cortical dysplasia of Taylor