Dong Seok Kim

Brain somatic mutations in MTOR cause focal cortical dysplasia type II leading to intractable epilepsy

Focal cortical dysplasia type II (FCDII) is a sporadic developmental malformation of the cerebral cortex characterized by dysmorphic neurons, dyslamination and medically refractory epilepsy. It has been hypothesized that FCD is caused by somatic mutations in affected regions. Here, we used deep whole-exome sequencing (read depth, 412–668×) validated by site-specific amplicon sequencing (100–347,499×) in paired brain-blood DNA from four subjects with FCDII and uncovered a de novo brain somatic mutation, mechanistic target of rapamycin (MTOR) c.7280T>C (p.Leu2427Pro) in two subjects. Deep sequencing of the MTOR gene in an additional 73 subjects with FCDII using hybrid capture and PCR amplicon sequencing identified eight different somatic missense mutations found in multiple brain tissue samples of ten subjects. The identified mutations accounted for 15.6% of all subjects with FCDII studied (12 of 77). The identified mutations induced the hyperactivation of mTOR kinase. Focal cortical expression of mutant MTOR by in utero electroporation in mice was sufficient to disrupt neuronal migration and cause spontaneous seizures and cytomegalic neurons. Inhibition of mTOR with rapamycin suppressed cytomegalic neurons and epileptic seizures. This study provides, to our knowledge, the first evidence that brain somatic activating mutations in MTOR cause FCD and identifies mTOR as a treatment target for intractable epilepsy in FCD.

CD40 ligand shedding is regulated by interaction between matrix metalloproteinase‐2 and platelet integrin αIIbβ3

Summary.  Background: CD40 ligand (CD40L, CD154) in the circulatory system is mainly contained in platelets, and surface‐expressed CD40L on activated platelets is subsequently cleaved by proteolytic activity to generate soluble CD40L (sCD40L). However, the enzyme responsible for the shedding of CD40L in activated platelets has not been clearly identified yet. We have recently found that molecular interaction of matrix metalloproteinase‐2 (MMP‐2) with integrin αIIbβ3 is required for the enhancement of platelet activation. Objectives: To elucidate the biochemical mechanism of MMP‐2‐associated sCD40L release. Methods: Localization of MMP‐2 and CD40L in platelets was analyzed by flow cytometry and fluorescence microscopy. The release of sCD40L from activated platelets was measured by enzyme‐linked immunosorbent assay. MMP‐2 binding to αIIbβ3 was analyzed by immunoprecipitation and western blotting. Recombinant hemopexin‐like domain and MMP‐2‐specific inhibitor were used to characterize the nature of MMP‐2 binding and catalytic activity. Results: It was revealed that interaction of MMP‐2 with αIIbβ3 is required for effective production of sCD40L in activated human platelets. Platelet activation and release of sCD40L were significantly affected by inhibition of platelet‐derived MMP‐2 activity or by inhibition of binding between the enzyme and the integrin. It was also found in platelet‐rich plasma that MMP‐2 activity is responsible for generating sCD40L. Conclusions: The results presented here strongly suggest that MMP‐2 interacts with αIIbβ3 to regulate the shedding of CD40L exposed on the surfaces of activated human platelets.

Treatment modality for intractable epilepsy in hypothalamic hamartomatous lesions

OBJECTIVEHypothalamic hamartomas (HHs) are often associated with early-onset gelastic seizures, thus configuring a well recognized and usually severe case of childhood epilepsy syndrome. We present a treatment modality for intractable epilepsy in hypothalamic hamartomatous lesions. METHODSThis study presents 14 patients with medically refractory seizure associated with HHs treated between 1995 and 2005. The HHs were diagnosed on the basis of magnetic resonance imaging, except in the case of one patient in whom hamartoma was confirmed histologically. There were seven boys and seven girls in this study. The most frequent clinical presentations were seizures. To identify the epileptic focus, we performed comprehensive epilepsy investigations, including electroencephalographic recording using a depth electrode into the hamartoma. RESULTSTo control the seizure, we performed surgical resection in one patient, gamma knife radiosurgery in four patients, and endoscopic disconnection in 11 patients. Seizure outcome was scored according to Engels classification throughout a mean follow-up period of 27.4 months (range, 3–54 mo). Of the 11 patients who underwent endoscopic disconnection, six were seizure-free immediately after surgery. Two patients were already diagnosed as having an HH and underwent gamma knife radiosurgery, but seizure control was not achieved. Their gelastic seizure disappeared after endoscopic disconnection. CONCLUSIONWe confirmed that HHs are intrinsically epileptogenic. Therefore, we suggest that HH-related seizures may be controlled by blocking the seizure propagation from epileptogenic HHs through simple disconnection, regardless of the treatment modality, and the endoscopic disconnection of HHs is safer and more effective than other modalities.

MMP‐2 regulates human platelet activation by interacting with integrin αIIbβ3

Summary.  Background: Human platelets contain matrix metalloproteinases (MMPs) that are secreted during platelet activation. Platelet MMPs have been implicated in the regulation of cellular activation and aggregation. Although the proaggregatory effect of MMP‐2 has been demonstrated, the functional mechanism is not clearly understood. Objectives: This work was carried out in order to elucidate the biochemical mechanism of MMP‐2‐associated platelet activation and aggregation. Methods: MMP‐2 binding to the platelet surface was analyzed by flow cytometry. The cell surface target of MMP‐2 was identified in thrombin receptor‐activating peptide‐stimulated platelets by immunoprecipitation, Western blotting and fluorescence microscopy. A recombinant hemopexin‐like domain was used to characterize the nature of MMP‐2 binding to the platelet surface. The functional significance of MMP‐2 in platelet activation was investigated by quantitative measurements of the activation markers P‐selectin (CD62P) and active αIIbβ3. The role of MMP‐2 in platelet aggregation was analyzed with an aggregometer. Results: ProMMP‐2 binds to integrin αIIbβ3 in stimulated platelets in which proMMP‐2 is converted into MMP‐2. Fibrinogen was able to replace the αIIbβ3‐bound MMP‐2. The molecular interaction of MMP‐2 and integrin αIIbβ3 was abrogated by the recombinant human hemopexin‐like domain of MMP‐2, leading to reduced cell surface expression of activation markers CD62P and active αIIbβ3, and resulting in suppressed platelet aggregation. Conclusion: This work clearly demonstrates that platelet activation and aggregation is regulated by MMP‐2 that specifically interacts with integrin αIIbβ3. The C‐terminal hemopexin‐like domain of MMP‐2 is an essential element for binding to αIIbβ3.

Low-dose craniospinal irradiation as a definitive treatment for intracranial germinoma

PURPOSE To determine the optimal radiotherapy (RT) dose and volume for treatment of intracranial germinoma. MATERIALS AND METHODS Eighty-one intracranial germinoma patients (33 pathologically-verified; 48 presumed by radiosensitivity testing) treated with RT alone between 1971 and 2002 were analyzed. The RT volume varied from focal (13) to whole brain (8), or to the entire neuraxis (60). All the cases after 1982 received craniospinal irradiation (CSI). Radiation dose was reduced gradually during the study period from 59 to 39.3 Gy for primary tumors, and from 34.2 to 19.5 Gy for the neuraxis. The median follow-up time was 120 months (48-260 months). RESULTS Five- and ten-year relapse-free survival rates were 98.8% and 94.1%, respectively. All the recurrences occurred in the patients who received local (4/13) or whole brain RT (1/8). None of the patients who received CSI suffered from a recurrence. Forty-six patients received 45 Gy or less to the primary site and 22 patients received less than 20 Gy to the spinal axis. CONCLUSION Low-dose CSI-based RT should remain the standard treatment for intracranial germinoma. The RT dose can be reduced to 39.3 Gy for primary tumor sites and to 19.5 Gy for the spinal axis.

Somatic Mutations in TSC1 and TSC2 Cause Focal Cortical Dysplasia

Focal cortical dysplasia (FCD) is a major cause of the sporadic form of intractable focal epilepsies that require surgical treatment. It has recently been reported that brain somatic mutations in MTOR account for 15%-25% of FCD type II (FCDII), characterized by cortical dyslamination and dysmorphic neurons. However, the genetic etiologies of FCDII-affected individuals who lack the MTOR mutation remain unclear. Here, we performed deep hybrid capture and amplicon sequencing (read depth of 100×-20,012×) of five important mTOR pathway genes-PIK3CA, PIK3R2, AKT3, TSC1, and TSC2-by using paired brain and saliva samples from 40 FCDII individuals negative for MTOR mutations. We found that 5 of 40 individuals (12.5%) had brain somatic mutations in TSC1 (c.64C>T [p.Arg22Trp] and c.610C>T [p.Arg204Cys]) and TSC2 (c.4639G>A [p.Val1547Ile]), and these results were reproducible on two different sequencing platforms. All identified mutations induced hyperactivation of the mTOR pathway by disrupting the formation or function of the TSC1-TSC2 complex. Furthermore, in utero CRISPR-Cas9-mediated genome editing of Tsc1 or Tsc2 induced the development of spontaneous behavioral seizures, as well as cytomegalic neurons and cortical dyslamination. These results show that brain somatic mutations in TSC1 and TSC2 cause FCD and that in utero application of the CRISPR-Cas9 system is useful for generating neurodevelopmental disease models of somatic mutations in the brain.

Outcomes of epilepsy surgery in childhood-onset epileptic encephalopathy

PURPOSE to evaluate the outcomes and role of epilepsy surgery in children with intractable epileptic encephalopathy (EE). METHODS ninety-five children (64 boys, 31 girls) with intractable EE were treated by epilepsy surgery at Severance Childrens Hospital from 2003 to 2008. Surgical treatments included lobar resection, hemispherotomy and corpus callosotomy (CC). Seventy-six children were Lennox-Gastaut syndrome (LGS), and 19 had West syndrome. RESULTS of the 76 patients with LGS, CC was performed in 37 patients (48.7%), lobar resection in 29 (38.2%) and hemispherotomy in 10 (13.2%). Of the 19 patients with West syndrome, respective surgery was performed in 15 patients (78.9%) and CC in 4 (21.1%). Of the patients receiving respective surgery, Engels class I outcomes were achieved for 24 of 39 (61.5%) of LGS patients, and for 9 of 15 (60.0%) of West syndrome. Malformations of cortical development were commonly observed, appearing in 73.5% (36/49). In neuropsychiatric tests, 19 of 27 with LGS demonstrated improvement in postoperative cognitive function. More significant intellectual improvement correlated well with shorter epilepsy duration, good seizure outcomes, and decreased number of antiepileptic drugs. CONCLUSIONS epilepsy surgery should be considered in treating childhood intractable EE with expectation of improvement of both seizure and cognitive outcomes, even in cases of LGS.

Uncovered primary seizure foci in Lennox–Gastaut syndrome after corpus callosotomy

PURPOSE Corpus callosotomy (CC) is a palliative surgical procedure to control atonic, tonic, or generalized tonic-clonic seizure in Lennox-Gastaut syndrome (LGS). Here, we report patients with LGS who underwent resective surgery, following CC better delineating the presumed seizure foci localized in one hemisphere. METHODS We retrospectively reviewed seven patients with LGS who underwent CC and subsequent cortical resection. The median follow-up duration after lobectomy was 20 months (range, 15-54 months) and three patients had follow-up periods over 24 months. The findings of video electroencephalography (EEG) monitoring, structural and functional neuroimagings were compared between pre- and post-CC. RESULTS Four patients had Engel class I and one patient had Engel class II outcomes following cortical resection; post-CC, compared to pre-CC, showed better localized ictal/interictal epileptiform discharges in the unilateral frontal area in two patients, in the unilateral parieto-temporo-occipital areas in one patient and in the unilateral fronto-temporal areas in the remaining two patients. Two patients had Engel Class III outcome following cortical resection; post-CC EEG continued to show multifocal epileptiform discharges but predominantly arising from a unilateral frontal area. Following CC, positron emission tomography showed localized glucose hypometabolism of which location was concordant with post-CC EEG abnormalities in all patient. Similarly, ictal/interictal single photon emission computed tomography also showed localized abnormalities concordant with post-CC EEG abnormalities in five of the six patients. Pathological assessment revealed cortical dysplasia in six patients, whereas no pathological abnormality was found in the remaining patient, who obtained Engel Class I outcome following cortical resection. CONCLUSION CC could change EEG findings, glucose metabolisms and cerebral blood flows, and it is sometimes helpful in delineating the primary seizure focus in patients with LGS.

Existence of glioma stroma mesenchymal stemlike cells in Korean glioma specimens

PurposeIt was presented that mesenchymal stem cells (MSCs) can be isolated from western glioma specimens. However, whether MSCs exist in glioma specimens of different ethnicities is unknown. To verify the existence of MSCs in an independent cohort, we undertook studies to isolate MSCs from a group of Korean patients. We hypothesized that cells resembling MSCs that were deemed mesenchymal stemlike cells (MSLCs) exist in an independent cohort of Korean gliomas.MethodsWe cultured fresh glioma specimens using the protocols used for culturing MSCs. The cultured cells were analyzed with fluorescence-activated cell sorting (FACS) for surface markers associated with MSCs. Cultured cells were exposed to mesenchymal differentiation conditions. To presume possible locations of MSLCs in the glioma, sections of glioma were analyzed by immunofluorescent labeling for CD105, CD31, and NG2.ResultsFrom nine of 31 glioma specimens, we isolated cells resembling MSCs, which were deemed Korean glioma stroma MSLCs (KGS-MSLCs). KGS-MSLCs were spindle shaped and adherent to plastic. KGS-MSLCs had similar surface markers to MSCs (CD105+, CD90+, CD73+, and CD45−). KGS-MSLCs were capable of mesenchymal differentiation and might be located around endothelial cells, pericytes, and in a disorganized perivascular area inside glioma stroma.ConclusionsWe found that cells resembling MSCs indeed exist in an independent cohort of glioma patients, as presented in western populations. We could presume that the possible location of KGS-MSLCs was in perivascular area or in glioma stroma that was a disorganized vascular niche. It might be possible that KGS-MSLCs could be one of constituent of stroma of glioma microenvironment.

Long-term prognosis of patients with Lennox–Gastaut syndrome in recent decades

PURPOSE We investigated long term prognosis of Lennox-Gastaut syndrome (LGS) with active application of recent advanced treatment modalities such as ketogenic diet (KD) or epilepsy surgery (ES). METHODS We retrospectively evaluated 68 patients with LGS, aged 18-35 years. We assessed seizure outcomes for a range of therapeutic modalities. Evolution of seizure types, EEG characteristics, cognition, ambulation, social outcomes and other clinical data were also evaluated. RESULTS For a mean follow-up duration of 19.3 years (range 8.3-32.5 years), finally sixteen patients (23.5%) were seizure-free. Of the 68 patients, 26 (38.2%) were treated solely with AEDs and six became seizure-free. The KD was administered to 19 patients, five patients maintained a seizure free state during the KD but only one patient was able to maintain a seizure free state by continuing on a modified Atkins diet. Focal resective surgery was performed in 15 patients, ten out of the 15 patients had Engel class I outcomes, but only three patients maintained seizure freedom to adults. CC was performed in 17 patients, and VNS was performed in 14 patients. These palliative procedures were also effective but had limitations to obtain and maintain seizure freedom to adults. Characteristic EEG features of diffuse slow spike-wave and generalized paroxysmal fast activity ceased in half of the patients. In cognitive terms, 94.7% of patients exhibited moderate to profound mental retardation. Only 39.7% of patients had intact independent daily living skills, and 25.4% could not walk, even with support. CONCLUSIONS KD and epilepsy surgery were effective for seizure control, but they did not necessarily lead to the maintenance of a seizure-free state. LGS remains a form of intractable epilepsy despite the application of recent advanced treatment modalities.