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

[Author] 
Ja Hye Kim, MD, PhD,1,2 Ji-Hyung Park, MD,1 Junehawk Lee, PhD,3 Jung Woo Park, PhD,3Hyun Jung Kim, MD, PhD,1,4 Won Seok Chang, MD, PhD,5,6 Dong-Seok Kim, MD, PhD,5Young Seok Ju, MD, PhD,1,7 Eleonora Aronica, PhD, 8,9 and Jeong Ho Lee, MD, PhD 1,10

1 Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic ofKorea;
2 Department of Pediatrics, Asan Medical Center Children’s Hospital, University of Ulsan College of Medicine, Seoul, Republic of Korea;
3 Center forSupercomputing Applications, National Institute of Supercomputing and Networking, Korea Institute of Science and Technology Information, Daejeon,Republic of Korea;
4 Department of Anatomy, Korea University College of Medicine, Seoul, Republic of Korea;
5 Department of Neurosurgery, YonseiUniversity College of Medicine, Seoul, Republic of Korea;
6 Brain Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea;
7 Genome Insight Inc., Daejeon, Republic of Korea;
8 Department of Pathology, Amsterdam UMC, University of Amsterdam, Amsterdam Neuroscience,Amsterdam, the Netherlands;
9 Stichting Epilepsie Instellingen Nederland, Heemstede, The Netherlands; and
10 SoVarGen Inc., Daejeon, Republic of Korea

[Journal] 

Annals of Neurology Volume93, Issue6, June 2023, Pages 1082-1093
 

[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