Screening and identification of novel candidate biomarkers of focal cortical dysplasia type II via bioinformatics analysis.

PURPOSE: Focal cortical dysplasia (FCD) is the most common developmental malformation that causes refractory epilepsy. FCD II is a common neuropathological finding in tissues resected therapeutically from patients with drug-resistant epilepsy. However, its molecular genetic etiology remains unclear. This study aimed to identify potential molecular markers of FCD II using bioinformatics analysis. METHODS: We downloaded two datasets for FCD II from the Gene Expression Omnibus data repository. Differentially expressed genes (DEGs) between FCD II and normal brain tissues were identified, and functional enrichment analysis was performed. A protein-protein interaction network was constructed, and hub genes were identified from the DEGs. The hub gene expression was validated using WB in vitro. IHC staining was performed to verify the feasibility of the target molecular markers identified in the bioinformatics analysis. RESULTS: One hundred sixty-seven common DEGs were identified between the datasets. The GO and KEGG analyses showed that variations were prominently enriched in some functions associated with gene expression. Five hub genes (i.e., FANCI, FANCA, BRCA2, RAD18, and KEAP1) were identified. Western blotting confirmed that all hub gene expressions were higher in the FCD II tissue than in the normal brain tissue. IHC staining showed that the FANCI expression significantly increased in the FCD II tissue. CONCLUSION: There are DEGs between FCD II and normal brain tissues, which may be considered biomarkers for FCD II, along with FANCI. The DEGs and hub genes identified in the bioinformatics analysis could serve as candidate targets for diagnosing and treating FCD II.

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

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 Patient 1. SIGNIFICANCE: Seven PDSVs were identified in FCDII lesions in six of 17 children. Five variant genes had not been previously associated with cortical malformations. We demonstrated that the IRS1 variant led to mTOR hyperactivation in vitro. Although functional experiments are needed, the results provide evidence for novel candidate genes in the pathogenesis of FCDII.

Remote MEG dipoles in focal cortical dysplasia at bottom of sulcus.

OBJECTIVE: To investigate whether the magnetoencephalography (MEG) single moving dipole (SMD) method could delineate the epileptic zone of focal cortical dysplasia (FCD) at the bottom of sulcus (FCDB). METHODS: We retrospectively analyzed 17 children (11 male; mean age 8.8 years, range 3-17 years) with FCD type II who underwent epilepsy surgery. We compared spatial congruence between the following: (1) MEG cluster and FCDB and (2) MEG cluster and FCD at the brain surface (FCDS). We measured the volume and depth of magnetic resonance imaging (MRI)-visible lesions to investigate whether they affect spatial congruence between MEG cluster and MRI-visible lesion. RESULTS: Eight children had FCDB and the other nine children had FCDS. The volume of MRI-visible lesions for FCDB ranged from 1,632 to 4,707 mm(3) (mean ± standard deviation [SD] 3,095 ± 1,211 mm(3) ). The depth of FCDB ranged from 19 to 33 mm (mean ± SD 26 ± 4 mm). The volume of MRI-visible lesion for FCDS ranged from 2,375 to 57,331 mm(3) (15,470 ± 18,455 mm(3) ). There was a tendency for a smaller volume of MRI-visible lesion for FCDB, relative to FCDS(p = 0.079). In FCDB, six children showed clusters of MEG dipoles and two children showed scattered MEG dipoles for interictal spikes. The spatial congruence between the MEG result and FCDB was partially overlapping in four children and discordant in another four children. In FCDS, eight children had MEG cluster and one child had MEG scatter alone. The spatial congruence between MEG result and FCDS was overlapping in eight of nine children (fully two; partially six) and discordant in one of nine children. Fifteen children (88%; FCDB eight; FCDS seven) became seizure-free after resective surgery. MEG spike dipole resection ratio in the cluster ranged from 4-100% (mean 67%) in 6 FCDB and 23-99% (mean 77%) in 8 FCDS. SIGNIFICANCE: The SMD method may drift MEG spike dipoles for FCDB. Lesionectomy can control seizures for four of eight patients in FCDB despite the remote MEG dipoles. The FCDB or FCDS partially overlapped with MEG cluster may have an extending/invisible epileptogenic zone consecutive to the MRI-visible lesion.

Intraoperative ECoG in bottom-of-the-sulcus syndrome using a novel flexible strip electrode.

The recording of epileptiform discharges from bottom-of-sulcus focal cortical dysplasia (BOSD) is often difficult during intraoperative electrocorticography (ECoG) due to the deep localization. We describe the use in this scenario of a new-generation electrode strip with high flexibility, easily adapted to cortical gyri and sulci. A right-handed 20-year-old male with drug-resistant focal epilepsy due to BOSD of the inferior frontal gyrus and daily focal aware seizures was evaluated for epilepsy surgery. Based on electroclinical and neuroimaging results, a focal cortectomy guided by ECoG was proposed. ECoG recordings were performed with new-generation cortical strips (Wise Cortical Strip; WCS®) and standard cortical strips. ECoG, performed on the convexity of the frontal cortical surface, recorded only sporadic spikes with both types of strips. Then, after microsurgical trans-sulcal dissection, WCS was molded along the sulcal surface of the suspected BOSD based on 3D-imaging reconstruction, showing continuous/subcontinuous 3-4-Hz rhythmic spike activity from the deepest electrode. Registration after resection of the BOSD did not show any epileptiform activity. Pathology showed dysmorphic neurons and gliosis. No surgical complications occurred. The patient is seizure-free after 12 months. This single case experience shows that highly flexible electrode strips with adaptability to cortical gyrations can identify IEDs originating from deep location and could therefore be useful in cases of bottom of the sulcus dysplasia.

Efficacy of sirolimus for epileptic seizures in childhood associated with focal cortical dysplasia type II.

OBJECTIVE: The efficacy of the mechanistic target of rapamycin inhibitor, sirolimus, was recently reported for patients more than 6 years of age by Kato et al. We evaluated the efficacy and safety of sirolimus in a 2-year-old patient with recurrent focal seizures with impaired consciousness after focal cortical dysplasia (FCD) type IIa resection. METHODS: The patient was a 2-year-old girl who had recurrent seizures after undergoing FCD resection at 4 months of age. The initial dose of sirolimus was 0.5 mg/day and was gradually increased using the trough blood concentration before oral administration as an index, and evaluation was performed at 92 weeks. RESULTS: The trough blood level of sirolimus was increased to 6.1 ng/mL and maintenance therapy was started at 40 weeks. Focal seizures with impairment of consciousness with tonic extension of the limbs decreased. No critically serious adverse events occurred. CONCLUSION: Sirolimus was effective against epileptic seizures from FCD type II even for a child under 5 years of age. There were no critically serious adverse events and administration could be continued.

Alteration of GABAergic neurons and abnormality of NKCC1/KCC2 in focal cortical dysplasia (FCD) type â ¡ lesions.

BACKGROUND: The current conclusions of molecular genetics still cannot satisfactorily explain the pathogenesis of focal cortical dysplasia (FCD) and the reason for drug resistance. The interneurons of GABA deserve attention. To observe the distribution and changes of GABAergic neurons and to explore the expression of cation chloride cotransporter NKCC1/KCC2 in focal cortical dysplasia (FCD) type II lesions is a highly significant job. METHODS: The expressions of GAD67(a marker of active GABAergic neuron), NKCC1 and KCC2 were detected by immunohistochemistry and immunohistochemistry double staining in 10 cases of FCD Ⅱa and 10 cases of FCD Ⅱb. The number of GAD67 positive neurons was counted, and the average absorbance (IA) of NKCC1 positive expression was measured, using Image Pro-Plus7.0 software. The data were statistically analyzed. RESULTS: The density of GABAergic neuron in focal dysplastic regions was significantly lower than that in the histologically "normal" cerebral cortex, regions from the same specimen (p < 0.0001, t-test). Compared to the NKCC1 staining intensity of neurons in the control group (measuring 1000 cells each), the IA value of dysmorphic neurons was significantly increased (p < 0.05, t'-test Cochran & Cox method). Intracytoplasmic concentration of KCC2 was evident in dysmorphic neurons but not in the other mature neurons. Most of the balloon cells were negative for NKCC1, except for few balloon cells showing sparse colored particles. The expression of KCC2 was negative in all balloon cells. CONCLUSIONS: The changes in the expression of NKCC1 and KCC2 may indicate that dysmorphic neurons were in a state similar to that of immature neurons. This state may be related to the abnormal electrophysiology of epilepsy. The difference between the number of GAD67 positive cells in the lesion site and the remote site of the same case may be an evaluation index of the effectiveness of surgery.

Localized focal cortical dysplasia type II: seizure freedom with lesionectomy guided by MRI and FDG-PET.

OBJECTIVE: The authors perform thorough, noninvasive presurgical evaluations for intractable epilepsy at their center and avoid unnecessary intracranial EEG when possible. The purpose of this study was to clarify the appropriateness of their lesion-oriented surgical strategy for localized focal cortical dysplasia (FCD) type II. METHODS: Fifty-one patients with pathologically proven localized FCD type II who were followed for at least 1 year after surgery were included. Patients with FCD type II with lobar or multilobar distribution were excluded. The results of presurgical evaluations, including thin-slice 3-T MRI, FDG-PET, and ictal SPECT, as well as surgical procedures and postoperative seizure and functional outcomes, were examined retrospectively. RESULTS: MRI was positive in 46 (90%) of 51 patients, and FDG-PET revealed localized hypo- or hypermetabolism in 47 (92%) of 51 patients. Ictal SPECT revealed concordant hyperperfusion in 37 of 42 patients examined. Intracranial EEG was used in only 13 patients (25%), including 5 with negative MRI results and 4 with subtle MRI findings. Of the 15 patients with FCD in the vicinity of eloquent (sensorimotor and language) areas, intracranial EEG was used in 4. Lesionectomy was performed in all 51 patients. Intraoperative electrocorticography (ECoG) was performed in 8 patients, but the findings were not used to tailor the extent of resection. Postoperative seizure outcomes were Engel class I in 47 patients (92%) and Ia in 45 (88%). In the 15 patients with FCD in the vicinity of eloquent areas, 13 (87%) achieved a class I outcome. Predictive factors for favorable seizure outcome were complete resection of the MRI lesion (p = 0.006) and frontal lobe surgery (p = 0.012). Postoperative neurological deficits were noted in only 4 (27%) of 15 patients with FCD in the vicinity of eloquent areas. All 5 MRI-negative patients achieved an Engel class I outcome. CONCLUSIONS: In most of the patients with localized FCD type II, MRI and/or FDG-PET detected the localized abnormality. Lesionectomy without intracranial EEG led to seizure freedom in most cases. Even when lesions were in the vicinity of eloquent areas, seizure and functional outcomes were favorable. Intraoperative ECoG may thus be unnecessary. Complete resection of the lesion is essential for favorable seizure outcome in MRI-positive patients. In MRI-negative patients, surgery with intracranial EEG guided by FDG-PET provided seizure-free outcomes.

Epilepsy in the mTORopathies: opportunities for precision medicine.

The mechanistic target of rapamycin signalling pathway serves as a ubiquitous regulator of cell metabolism, growth, proliferation and survival. The main cellular activity of the mechanistic target of rapamycin cascade funnels through mechanistic target of rapamycin complex 1, which is inhibited by rapamycin, a macrolide compound produced by the bacterium Streptomyces hygroscopicus. Pathogenic variants in genes encoding upstream regulators of mechanistic target of rapamycin complex 1 cause epilepsies and neurodevelopmental disorders. Tuberous sclerosis complex is a multisystem disorder caused by mutations in mechanistic target of rapamycin regulators TSC1 or TSC2, with prominent neurological manifestations including epilepsy, focal cortical dysplasia and neuropsychiatric disorders. Focal cortical dysplasia type II results from somatic brain mutations in mechanistic target of rapamycin pathway activators MTOR, AKT3, PIK3CA and RHEB and is a major cause of drug-resistant epilepsy. DEPDC5, NPRL2 and NPRL3 code for subunits of the GTPase-activating protein (GAP) activity towards Rags 1 complex (GATOR1), the principal amino acid-sensing regulator of mechanistic target of rapamycin complex 1. Germline pathogenic variants in GATOR1 genes cause non-lesional focal epilepsies and epilepsies associated with malformations of cortical development. Collectively, the mTORopathies are characterized by excessive mechanistic target of rapamycin pathway activation and drug-resistant epilepsy. In the first large-scale precision medicine trial in a genetically mediated epilepsy, everolimus (a synthetic analogue of rapamycin) was effective at reducing seizure frequency in people with tuberous sclerosis complex. Rapamycin reduced seizures in rodent models of DEPDC5-related epilepsy and focal cortical dysplasia type II. This review outlines a personalized medicine approach to the management of epilepsies in the mTORopathies. We advocate for early diagnostic sequencing of mechanistic target of rapamycin pathway genes in drug-resistant epilepsy, as identification of a pathogenic variant may point to an occult dysplasia in apparently non-lesional epilepsy or may uncover important prognostic information including, an increased risk of sudden unexpected death in epilepsy in the GATORopathies or favourable epilepsy surgery outcomes in focal cortical dysplasia type II due to somatic brain mutations. Lastly, we discuss the potential therapeutic application of mechanistic target of rapamycin inhibitors for drug-resistant seizures in GATOR1-related epilepsies and focal cortical dysplasia type II.

Clinical features and surgical outcomes in young children with focal cortical dysplasia type II.

AIMS: To investigate clinical characteristics and surgery outcomes of young children with focal cortical dysplasia (FCD) type II. METHODS: Young children (onset age ≤6 years) with FCDII who underwent epileptic surgery in Children Epilepsy Center of Peking University First Hospital in 2014-2018 were followed up for at least 6 months after surgery. RESULTS: One hundred and twelve children with FCDII were included, with median age of onset 0.9 years (0.01-5.9), who underwent surgery at 4.1 years old (0.8-16.2). Focal seizures were most frequent (90.2%) and epileptic spasms presented in 23 (20.5%) cases. Epileptic encephalopathy was not uncommon (12.5%), associated with earlier epilepsy onset and higher rate of bilateral onset on ictal EEG (OR = 0.213, 9.059; P = .041, .004). At the last follow-up, 88.4% achieved seizure-free. Before surgery, 49.1% showed moderate/severe developmental delay, associated with earlier seizure onset and higher rate of history of epileptic encephalopathy (OR = 0.740, 5.160, P = .023, .042). For 48 children with preoperatively moderate/severe developmental delay, DQ rank at 6 months postsurgery was improved in only four cases. CONCLUSION: For young children with FCDII, they tend to present with epileptic encephalopathies and show moderate/severe developmental delay before surgery. The seizure outcome was favorable after surgery. For children with preoperatively moderate/severe developmental delay, developmental outcome at 6 months after surgery was not satisfactory.

Small Lesion Size Is Associated with Sleep-Related Epilepsy in Focal Cortical Dysplasia Type II.

OBJECTIVE: To investigate the neuroimaging and clinical features associated with sleep-related epilepsy (SRE) in patients with focal cortical dysplasia (FCD) type II. METHODS: Patients with histopathologically proven FCD type II were included from three epilepsy centers. SRE was defined according to the video EEG findings and seizure history. Cortical surface reconstruction and volume calculation were performed using FreeSurfer. The lesions were manually delineated on T1 volumetric MRI using the ITK-SNAP software. The lesion volumes were normalized by the intracranial volume of each patient. The lesions were classified as small or large by placing a threshold based on quantitative (whether the lesion was detected on MRI report) and qualitative (volume) criteria. RESULTS: A total of 77 consecutive patients were included. Of them, 36 had SRE and 41 had non-SRE. An earlier age of epilepsy onset, high seizure frequency, regional interictal EEG findings, and favorable surgical outcome were characteristic in both groups. Small lesions were defined as those having a volume <3,217 mm3. In total, 60.9% of the patients with SRE (25/41) had small FCD lesion, which was significantly higher than the non-SRE group (9/34, 26.5%, p = 0.005). Small lesion size was the only predictor significantly associated with SRE in the overall type II group by multivariate analyses (p = 0.016). Although the proportion of patients who had frontal FCD and SRE was higher than non-frontal FCD (54.5 vs. 27.3%, p = 0.043), the relationship between SRE and lesion location was not confirmed by multivariate analysis. Thalamic volume and seizure semiology were not statistically different between the SRE and non-SRE group. The significant association between lesion size and SRE was reproducible in type IIb and IIa subgroups. SIGNIFICANCE: SRE is common in patients with FCD type II. Small FCD type II lesions are significantly associated with SRE. Although our findings cannot be applied to the entire spectrum of SRE, potential existence of small FCD lesions should be considered when evaluating patients with SRE, and utilization of all other supportive electroclinical information for lesion detection is highly desirable.

Notch signaling in human iPS-derived neuronal progenitor lines from Focal Cortical Dysplasia patients.

Focal Cortical Dysplasia (FCD), a common type of Malformations of cortical development, may result from an early disturbance in the migration and final arrangement of the cortical architecture of immature neurons. FCD type II is now known to be due to a post-zygotic somatic mutation that involves the mTOR and AKT pathways. The aim of the present study was to investigate the possible differences in neurogenesis and neurodifferentiation of iPSCs (induced pluripotent stem cells) from fibroblasts of individuals affected by FCD type II (2) and normal individuals (2). iPSCs were generated from skin fibroblasts of FCD individuals and healthy individuals. The reprogramming was done through the fibroblasts exposure to viral vectors containing the OCT4, KLF4, SOX2, and c-MYC genes and the clones were characterized by immunohistochemistry. iPSCs were neurodifferentiated and analyzed at the 14th, 22nd and 35th days. We also analyzed the cerebral cortex tissue, fibroblasts and iPSCs cells from the individuals. Through qRT-PCR, the expression of 4 genes involved in Notch signaling process were quantified. In general, individuals with dysplasia presented increase and decrease in the relative quantification in the most genes analyzed compared to control individuals in all processes and study groups. We suggest that, during embryonic neurogenesis, the neural precursor cells of FCD type II individuals present increase and decrease in gene expression in the Notch signaling pathway causing cortical formation disorders and can be seen as a candidate for the developmental changes observed in the cerebral cortex of individuals with FCD type II. This altered gene expression may be related to brain formation with dysplasia.

Advanced dynamic statistical parametric mapping with MEG in localizing epileptogenicity of the bottom of sulcus dysplasia.

OBJECTIVE: To investigate whether advanced dynamic statistical parametric mapping (AdSPM) using magnetoencephalography (MEG) can better localize focal cortical dysplasia at bottom of sulcus (FCDB). METHODS: We analyzed 15 children with diagnosis of FCDB in surgical specimen and 3 T MRI by using MEG. Using AdSPM, we analyzed a ±50 ms epoch relative to each single moving dipole (SMD) and applied summation technique to estimate the source activity. The most active area in AdSPM was defined as the location of AdSPM spike source. We compared spatial congruence between MRI-visible FCDB and (1) dipole cluster in SMD method; and (2) AdSPM spike source. RESULTS: AdSPM localized FCDB in 12 (80%) of 15 children whereas dipole cluster localized six (40%). AdSPM spike source was concordant within seizure onset zone in nine (82%) of 11 children with intracranial video EEG. Eleven children with resective surgery achieved seizure freedom with follow-up period of 1.9 ±â€¯1.5 years. Ten (91%) of them had an AdSPM spike source in the resection area. CONCLUSION: AdSPM can noninvasively and neurophysiologically localize epileptogenic FCDB, whether it overlaps with the dipole cluster or not. SIGNIFICANCE: This is the first study to localize epileptogenic FCDB using MEG.

Clinical and immunohistochemical characteristics of type II and type I focal cortical dysplasia.

Focal cortical dysplasia (FCD) II and I are major causes for drug-resistant epilepsy. In order to gain insight into the possible correlations between FCD II and FCD I, different clinical characteristics and immunohistochemical expression characteristics in FCD I and II were analyzed. The median age of onset and duration of epilepsy in FCD I and FCD II patients were 2.1 years and 5.3 years vs 2.4 years and 4.5 years. Therefore, the median age of onset and duration of epilepsy were similar in the two groups. Pathological lesions were predominantly located in frontal lobe in FCD II and temporal in FCD I. Significantly more signal abnormalities in FLAIR and T2 images were demonstrated in FCD II than FCD I. The rate of satisfied seizure outcome was relative higher in FCDII patients (95.12%) than that in FCDI group (84.6%). Furthermore, we detected expressions of progenitor cell proteins and the mammalian target of rapamycin (mTOR) cascade activation protein in FCDs. Results showed that sex-determiningregion Y-box 2(SOX2) , Kruppel-likefactor 4 (KLF4) and phospho-S6 ribosomal proteins (ser240/244 or ser235/236) were expressed in FCDII group but not in FCD I. Overall, this study unveils FCD I and II exhibit distinct clinical and immunohistochemical expression characteristics, revealing different pathogenic mechanisms.