Identification of genetic characteristics in pediatric epilepsy with focal cortical dysplasia type 2 using deep whole‐exome sequencing

Abstract Background Focal cortical dysplasia type 2 (FCD2) is a malformation of cortical development that constitutes a common cause of pediatric focal epilepsy. Germline or somatic variants in the mammalian target of rapamycin (mTOR) signaling pathway genes are the pathogenesis of FCD2. Objective In this study, whole‐exome deep sequencing was performed on dysplastic cortex from focal epilepsy in children to explore genetic characteristics in FCD2. Methods Resected core lesions of FCD2 were confirmed by pathology, and peripheral blood was collected from 11 patients. Deep whole‐exome sequencing (>500X) was performed on derived genomic DNA, germline, or somatic variants in brain‐specific genes were analyzed and identified. Results In 11 patients, a heterozygous likely pathogenic germline variant of DEPDC5 was identified in one case, while somatic variants were found in four brain samples. The frequencies of the somatic variant allele were 2.52%–5.12%. Somatic variants in AKT3, TSC2, and MTOR (mTOR signaling pathway genes) were found in three samples. Besides, one somatic variant was detected in MED12 which has not been reported to associate with FCD2. Conclusion Our study expanded the variant spectrum in the mTOR‐GATOR pathway, and also detected a somatic variant in MED12 which was potentially associated with FCD 2.


| INTRODUCTION
Focal cortical dysplasia (FCD) is the most common structural cause of intractable focal epilepsy in children (Gaitanis & Donahue, 2013). According to the International League Against Epilepsy (ILAE) classification, FCDs are categorized into type 1, type 2, and type 3 at pathology level (Scheffer et al., 2017). Characteristics of FCD2 include cortical dyslamination and dysmorphic neurons (DNs) without (FCD 2A) or with balloon cells (FCD 2B) (Blumcke et al., 2011). Compared with other types of FCDs, FCD2 occurred more common in children and exhibited more severe seizures. Recent studies revealed somatic variants of FCD2 in several genes associated with mammalian target of rapamycin ( (Baldassari et al., 2019;Jansen et al., 2015;Moller et al., 2016;Nakashima et al., 2015;Zhang et al., 2020;Zhao et al., 2019). Up to now, genetic exploration of FCD2 remains limited, here we aimed to uncover somatic variants in FCD 2-related genes by using deep whole exome sequencing of FCD core lesions and blood from 11 pediatric focal epilepsy.

| Clinical features
Detailed clinical features were listed in Table 1. Totally, 11 patients retrospectively analyzed who were diagnosed with focal epilepsy and had performed resective surgery from 2019 to 2021 at pediatric epilepsy center in the Children's Hospital of Fudan University. Imaging features of FCD2 including cortical thickness, signal change, gray-white blurring, and transmantle sign were collected via presurgical brain magnetic resonance imaging (MRI), subtype (FCD2A or FCD2B) was confirmed by postsurgical pathology. All FCD lesions were localized by video electroencephalography monitoring and fluorodeoxyglucose PET.

| Samples collection
The core region of FCD tissues was collected and immediately stored at −80°C at the time of surgery. Peripheral blood samples of patients were collected during hospitalization. The portion of the samples was subjected to hematoxylin-eosin (H&E) staining to confirm postsurgical pathology for FCD2. The study was approved by the Institutional Review Board (IRB) ethics committee of the Children's Hospital of Fudan University.

| Histopathology
H&E staining and light microscopy were used by neuropathologists to evaluate histopathological diagnosis of resected brain tissues.

| DNA extraction, sequencing, and data analysis
Frozen tissues were lysed and homogenized using mir-Vana kit lysis buffer (Ambion), a micropestle, and QIAshredder columns (Qiagen). DNA was isolated using TIANamp Genomic DNA Kit (Tiangen) following the official instruction. DNA integrity was confirmed by agarose gel electrophoresis, and concentration was determined by Qubit 2.0 fluorometer (Invitrogen).
The exon region was captured from peripheral blood and tissue DNA using xGen Exome Research Panel probes (IDT, USA) following the manufacturer's recommendations, then sequenced on MGISEQ 2000 platform (pairedend 150). The average depth per sample was larger than 500 coverage. Sequence alignment was performed by a Burrows-Wheeler algorithm (BWA), and variant calling was performed by Genome Analysis Tool Kit (GATK v4) best practices (https://softw are.broad insti tute.org/ gatk/bestp racti ces/) from the Broad Institute. Variants were annotated via ANNOVAR (http://www.openb ioinf ormat ics.org/annov ar/). Candidate variants were picked up in exonic and splicing regions with a minor allele frequency of ≤0.005 in SNP database (ExAC_EAS, ExAC_ ALL, 1000Genomes, gnomAD). Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping version 2 (PolyPhen-2) were utilized to predict the deleteriousness of amino acid substitution for the protein. Pathogenic classification was evaluated following the ACMG (American College of Medical Genetics and Genomics) guidelines.

| Clinical features of patients with FCD2
Eleven pediatric patients with FCD2 who underwent surgical resection were explored, including five cases with FCD 2A and six cases with FCD2B. Among them, six patients were males and five were females. The age at surgery ranged from 1.75 to 14 years old (median age 3.5-4.25), the age at seizure onset was from 0.75 to 12 years (median age 1.5) and the duration of seizures was from 0.25 to 11 years (median year 0.75-1.25). Nine patients exhibited focal seizures, one exhibited epileptic spasm, and one with focal seizure and epileptic spasm simultaneously. One lesion localized at left temporal lobe, one at right temporo-parieto-occipital lobe, one at right insular, two at left frontal lobe, and six at right frontal lobe (Table 1). MRI and histopathology of FCD2 were shown in Figures 1 and 2, respectively.

| DISCUSSION
To date, several studies suggest that FCDs are malformations of cortical development caused by germline or somatic variants in genes regulating proliferation, differentiation, ion channel, and cell signaling pathways (Iffland & Crino, 2017). In particular, FCD 2 is associated with genetic variants in the crucial mTOR-GATOR pathway. Due to the limitations of most studies using conventional WES, deep targeted gene sequencing (>500×) (Baldassari et al., 2019;Jansen et al., 2015;Kumari et al., 2020;Moller et al., 2016;Sim et al., 2019;Zhang et al., 2020), or deep WES for few FCD 2 patients (Bennett et al., 2022;Jha et al., 2022;Nakashima et al., 2015;Zhao et al., 2019), the associated causative genes for FCD 2 may not be fully discovered. In this study, for 11 FCD patients with core Recent studies have demonstrated that the somatic variants of genes within the PI3K-AKT-mTOR/TSC signaling pathway were the most important pathogenesis of FCD 2, such as PIK3CA, AKT3, MTOR, TSC1/2, and RHEB. Therefore, more than 60 cases with FCD 2 have been reported to have pathogenic or potentially deleterious somatic variants in the above genes, and the MTOR and TSC1/2 somatic variants were the most common, accounting for about 75%. The variant allele frequencies (VAFs) in FCD 2 ranged from 0.25% to 15.6%, and more than 15% were TSC1/2 somatic variants, VAFs ranged from 1.4% to 50.1% (Baldassari et al., 2019;Bennett et al., 2022;Blumcke et al., 2021;Jansen et al., 2015;Jha et al., 2022;Kumari et al., 2020;Lee et al., 2020;Moller et al., 2016;Nakashima et al., 2015;Sim et al., 2019;Zhang et al., 2020;Zhao et al., 2019). In our study, we found that cases 2 and 4 with FCD 2A had somatic variants in AKT3 F I G U R E 1 (a-k) MRI scan (T2 FLAIR sequences in axial or sagittal view) of 11 patients with FCD 2 (the order of English alphabetical corresponds to the number of case). Arrows indicate the location of FCD2 lesions.
DEPDC5 (DEP domain containing 5) combines with NPRL2 and NPRL3 to form the GATOR1 complex, which is a critical repressor of the mTOR pathway. Recently, several studies have identified germline and somatic variants in the DEPDC5 gene in FCD 2 patients (Baldassari et al., 2019;Blumcke et al., 2021;Gaitanis & Donahue, 2013;Lee et al., 2020;Ribierre et al., 2018;Sim et al., 2019). In our study, a likely pathogenic germline DEPDC5 variant (c.2875delA, frameshift) was found in case 5 with FCD 2A. It is unclear how germline variants in the DEPDC5 gene cause FCD, while possible mechanism of a second-hit somatic variant has revealed the development of DNs Ribierre et al., 2018). However, the second hit of the DEPDC5 somatic variant is unable to be detected in our case, similar to the results of some studies using deep targeted sequencing (Blumcke et al., 2021;Lee et al., 2020). Therefore, the exact correlation between FCD and DEPDC5 second-hit somatic variant requires to be explored by new research methods, such as single-cell sequencing for DNs.
MED12 is a subunit of the mediator complex which regulates cell growth, development, and differentiation, plays a crucial role in neural development. Hemizygous pathogenic missense variants in MED12 located on chromosome Xq13.1 cause three different but overlapping X-linked neurodevelopmental syndrome, such as FG syndrome, Lujan-Fryns syndrome, and Ohdo syndrome. The common phenotypes of the nervous system in these three syndromes were characterized by mental retardation, relative macrocephaly, and seizures (Polla et al., 2021;Srivastava & Kulshreshtha, 2021).
As part of the Mediator kinase module, MED12 is involved in multiple signaling pathways leading to transcriptional repression or activation, such as Wnt, mTOR pathway, etc. (Srivastava & Kulshreshtha, 2021). Several studies have demonstrated the Wnt pathway is critical for cellular determination during embryogenesis, and that downregulation of MED12 results in inactivation of the Wnt signaling in embryos, while inhibition of glycogen synthase kinase 3 (GSK3) in the Wnt pathway can phosphorylate TSC1 and TSC2 activate mTOR (Hermida et al., 2017;Inoki et al., 2006;Kim et al., 2006). Meanwhile, recent studies have shown that MED12 variants increase AKT expression in cell, resulting in simultaneous inhibition of GSK3β and activation of the mTOR pathway which might be associated with autophagy abrogation and cell proliferation (El Andaloussi et al., 2020). We identified a somatic missense variant of the MED12 gene in a male FCD 2A individual. Based on the above results, it is reasonable to speculate that MED12 is potentially pathogenic gene for FCD, and further research is necessary to confirm the relationship between MED12 and FCD.
In summary, we found four somatic and one germline variants including ATK3, MTOR, TSC2, MED12, and DEPDC5 in 5 of 11 FCD 2 pediatric individuals with epilepsy. Similar to previous studies of cortical malformations, we further demonstrated FCD 2 was associated with genetic variants in the crucial mTOR-GATOR pathway. MED12 leading to neurodevelopmental syndrome and being an upstream regulator of the mTOR pathway, however, somatic variant in MED12 has not been reported in FCD 2 patient before. Our findings suggest that the somatic variant in MED12 was potentially associated with FCD 2 and then further functional experiments are required to determine the role of MED12 in FCD 2 formation.

AUTHOR CONTRIBUTIONS
YX, RZH, and WM were the first author and co-first authors who performed data analysis and interpretation, and drafted the manuscript., XHW and YYM performed interpretation of clinical data. HL and YW conceived the idea for the study and supervised the study. BBW and YFZH is the corresponding author and conceived the idea for the study, designed the experiments, responsible for the analysis and interpretation of sequencing data and drafted and revised the manuscript content. All authors participated in the proofreading. The authors have read and approved the final manuscript.

ACKNOWLEDGMENTS
We would like to thank the patients and thier families who contributed to this study. We also thank Zuozhen Yang of CpherGene LLC for his assistance in genetic analysis.

FUNDING INFORMATION
This study was supported by Research Project of Shanghai Municipal Health Commission (201940351) and the science and technology commission of Shanghai (18411962000). The funding bodies played no direct role in the design of the study, the collection, analysis, or interpretation of data, or the writing of the manuscript. The cost of sequencing was borne by the two funding projects.

CONFLICT OF INTEREST
The authors declare that they have no competing interests.

ETHICS APPROVAL
The local ethics committee of Children's Hospital of Fudan University approved the study protocol.

CONSENT TO PARTICIPATE
Parents gave written informed consent for study participation.

CONSENT FOR PUBLICATION
Written informed consent for publication for identifying images or other personal or clinical details was obtained from both the patient's parents.