Somatic variants in new candidate genes identified in focal cortical dysplasia type II

Abstract

Objective: Focal cortical dysplasia type II (FCDII) is a malformation of cortex development commonly found in children with drug-resistant epilepsy. FCDII has been associated with somatic mutations in mammalian target of rapamycin (mTOR)-related pathway genes and an upregulation of mTOR. Somatic mutations were found in 10%-63% of FCDII samples; the frequency of the mutant allele was 0.93%-33.5%. This study aimed to find new candidate genes involved in FCDII. Methods: We collected resected FCD lesions, perilesional brain tissues, and peripheral blood from 17 children with pathologically confirmed FCDII. We performed whole exome sequencing and followed a set of screening and analysis strategies to identify potentially deleterious somatic variants (PDSVs) in brain-expressed genes.We performed site-specific amplicon sequencing to validate the results. We also performed an in vitro functional study on an IRS1 variant. Results: In six of 17 samples, we identified seven PDSVs in seven genes, including two frameshift variants and five missense variants. The frequencies of the variant allele were 1.29%-5.50%. The genes were MTOR, TSC2, IRS1, RAB6B, RALA, HTR6, and ZNF337. PDSVs in IRS1, RAB6B, ZNF337, RALA, and HTR6 had not been previously associated with FCD. In one lesion, two PDSVs were found in two genes. In a transfected cell line, we demonstrated that the c.1791dupG (identified in FCDII from Patient 1) led to a truncated IRS1 and significant mTOR hyperactivation compared to cells that carried wild-type IRS1. mTOR was also activated in FCDII tissue from

The Role of KRAS Mutations in Cortical Malformation and Epilepsy Surgery: A Novel Report of Nevus Sebaceous Syndrome and Review of the Literature

“…KRAS is involved in the MAPK pathways and could be related to cortical malformation because RAS/extracellular-signal-regulated kinase signaling pathways are potential activators of mTORC1 and mTORC2 upstream [ 77 ]. Recently, the possible association between somatic RAS-pathway variants and FCD type II has been highlighted [ 78 ].…”

Application of single cell genomics to focal epilepsies: A call to action

“…In order to understand whether a somatic variant contributes to disease in a cell‐autonomous manner or if it acts through disruption of complex cellular networks, it is important to know which cells carry the variant and how they differ from their genotypically normal counterparts. In the next phase of discovery that ensued, targeted sequencing of the affected brain tissue helped identify a large set of somatic variants, the majority of which were associated with MCD (21–33). This experimental approach has not been as effective for non‐lesional cases, particularly most adult‐onset focal epilepsies, although these cases are not as well studied.…”

Genomic and Epigenetic Advances in Focal Cortical Dysplasia Types I and II: A Scoping Review

“…32). A section of the resected brain specimen was formalin-fixed and paraffinembedded (FFPE) for the neuropathological classification in FCD type I, IIa, or IIb, and an adjacent block was immediately frozen in liquid nitrogen or stored at −80 • C for research purposes (Lim et al, 2015;Nakashima et al, 2015;Baldassari et al, 2019b;Zhao et al, 2019;Lee et al, 2020;Zhang et al, 2020).…”

“…The experimental design employed by various studies comprised an initial deep whole exome sequencing using matched samples of peripheral blood lymphocytes or saliva and surgically resected fresh-frozen or formalin-fixed paraffinembedded brain tissue, followed by bioinformatics analysis of the raw sequencing data and variant identification, with final validation using various targeted sequencing methods with high coverage (>100×) (Lim et al, 2015(Lim et al, , 2017Nakashima et al, 2015;Moller et al, 2016;Baldassari et al, 2019b;Sim et al, 2019;Zhao et al, 2019;Zhang et al, 2020). Subsequently, several studies performed in vitro functional analysis, with kinase assays and immunohistochemistry/immunoblotting for the detection of phosphorylated upstream (phospho-AKT) (Schick et al, 2006;Conti et al, 2015;Jansen et al, 2015;Mirzaa et al, 2016) or downstream (phospho-S6 ribosomal or 4EBP proteins) mediators of the mTOR signaling pathway in pathological brain samples and transfected heterologous mammalian cell lines (HEK293T) (Lim et al, 2015(Lim et al, , 2017Nakashima et al, 2015;Moller et al, 2016;Ribierre et al, 2018;Zhao et al, 2019;Zhang et al, 2020). These findings in human tissue led investigators to ascertain the pathogenicity of the detected brain mosaic variants in mouse models, through in utero electroporation combined, in some cases, with the CRISPR-Cas9 gene editing system (Lim et al, 2015(Lim et al, , 2017Ribierre et al, 2018;Zhao et al, 2019).…”

“…Brain-only somatic MTOR mutations accounted for 3.8-57.1% of patients with FCD II across different studies and represented the most commonly affected gene (Leventer et al, 2015;Lim et al, 2015;Nakashima et al, 2015;Mirzaa et al, 2016;Moller et al, 2016;D'Gama et al, 2017;Avansini et al, 2018;Baldassari et al, 2019b;Sim et al, 2019;Zhang et al, 2020). The variant allelic frequencies (VAF) detected in the brain-derived DNA were low, ranging from 0.25 to 15.67% for FCD IIa and from 0.74 to 10.6% for FCD IIb (Leventer et al, 2015;Lim et al, 2015;Nakashima et al, 2015;Mirzaa et al, 2016;Moller et al, 2016;D'Gama et al, 2017;Avansini et al, 2018;Baldassari et al, 2019b;Sim et al, 2019;Zhang et al, 2020). All MTOR somatic variants were missense, leading to a substitution of an amino acid in the protein.…”