Somatic mosaic deletions involving SCN1A cause Dravet syndrome.

Somatic mosaicism in single nucleotide variants of SCN1A is known to occur in a subset of parents of children with Dravet syndrome (DS). Here, we report recurrent somatic mosaic microdeletions involving SCN1A in children diagnosed with DS. Through the evaluation of 237 affected individuals with DS who did not show SCN1A or PCHD19 mutations in prior sequencing analyzes, we identified two children with mosaic microdeletions covering the entire SCN1A region. The allele frequency of the mosaic deletions estimated by multiplex ligation-dependent probe amplification and array comparative genomic hybridization was 25-40%, which was comparable to the mosaic ratio in lymphocytes and buccal mucosa cells observed by fluorescence in situ hybridization analysis. The minimal prevalence of SCN1A mosaic deletion is estimated to be 0.9% (95% confidence level: 0.11-3.11%) of DS with negative for SCN1A and PCDH19 mutations. This study reinforces the importance of somatic mosaicism caused by copy number variations in disease-causing genes, and provides an alternative spectrum of SCN1A mutations causative of DS. Somatic deletions in SCN1A should be considered in cases with DS when standard screenings for SCN1A mutations are apparently negative for mutations.

Immunological studies of cerebrospinal fluid from patients with CNS symptoms after human papillomavirus vaccination.

In 32 patients with prolonged central nervous system symptoms after human papillomavirus (HPV) vaccination, we measured conventional and immunological markers in cerebrospinal fluid (CSF) and compared with the levels in disease controls. Our studies revealed significantly decreased chloride and neuron-specific enolase (NSE) levels in CSF of patients with CNS symptoms after HPV vaccination compared to disease controls. IL-4, IL-13, and CD4(+) T cells increased significantly in patients, and IL-17 increased significantly from 12 to 24months after symptom onset. Chemokines (IL-8 and MCP-1) were also elevated, but CD8(+) T cells, PDGF-bb and IL-12 were reduced. Antibodies to GluN2B-NT2, GluN2B-CT and GluN1-NT increased significantly. These results suggest biological, mainly immunological, changes in the CSF of patients after HPV vaccination.

Antibodies against peptides of NMDA-type GluR in cerebrospinal fluid of patients with epileptic spasms.

OBJECTIVE: We investigated the contribution of antibodies against N-methyl-d-aspartate (NMDA)-type glutamate receptor (GluR) in cerebrospinal fluid (CSF) to the clinical features of patients with epileptic spasms (ES). METHODS: CSF samples were collected from 33 patients with ES with median (range) age 1.8 (0.2-8.5) years. Thirty patients without ES with 3.5 (0.5-7.0) years were also studied as disease controls. The CSF levels of antibodies against peptides of NMDA-type GluR subunits (GluN2B & GluN1) were measured by enzyme-linked immunosorbent assay. RESULTS: The levels of antibodies against the n-terminal of GluN2B (GluN2B-NT2), c-terminal of GluN2B (GluN2B-CT) and n-terminal of GluN1 (GluN1-NT), were significantly higher in patients with ES than in disease controls (p < 0.01, p < 0.01 & p = 0.03). Levels of antibodies to GluN2B-NT2 & CT were not related with ACTH therapy nor conventional CSF factors (cell counts, protein level, etc). Levels of antibodies to GluN2B-NT2 & CT showed evidence of correlation within a linear regression model with intervals from the onset to the examination of CSF until 25 months (p = 0.01 & p = 0.01). The correlation was significant in patients with unknown cause (p = 0.01). Five of 33 patients (four unknown cause & one chromosomal anomaly) had higher level of antibodies to GluN2B-NT2 exceeding mean + 1 SD of all ES patients, and they had poor motor (score 0) and cognitive outcomes (score 0 or 1). CONCLUSION: The CSF level of antibodies against GluN2B in ES patients with unknown cause was estimated to increase after onset. We hypothesize that some ES patients may have immune process after the onset of ES.

Three patients manifesting early infantile epileptic spasms associated with 2q24.3 microduplications.

BACKGROUND: Recent development of genetic analyses enabled us to reveal underlying genetic causes of the patients with epileptic encephalopathy in infancy. Mutations of voltage-gated sodium channel type I alpha subunit gene (SCN1A) are to be causally related with several phenotypes of epilepsy, generalized epilepsy with febrile seizure plus (GEFS+), Dravet syndrome, and other infantile epileptic encephalopathies. In addition to SCN1A, contiguous genes such as SCN2A and SCN3A in 2q24.3 are also reported to have contribution to epileptic seizures. Therefore, gene abnormality involving this region is reasonable to contribute to epilepsy manifestation. RESULTS: We encountered three patients with 2q24.3 microduplication diagnosed by Array comparative genomic hybridization array (aCGH). They developed partial seizures and epileptic spasms in their early infantile periods and showed remarkable developmental delay, although their seizures disappeared from 11 to 14 months of age. One of three patients had 2q24.3 microduplication which excludes SCN1A. Therefore, characteristics of epilepsy with 2q24.3 microduplication do not necessarily need duplication of SCN1A. This study suggested that 2q24.3 microduplication is one of the causes for early infantile epileptic spasms. Epileptic spasms associated with 2q24.3 microduplications may have better seizure outcome comparing with other etiologies.

Individualized phenytoin therapy for Japanese pediatric patients with epilepsy based on CYP2C9 and CYP2C19 genotypes.

BACKGROUND: The aims of this study were to identify the target dose of phenytoin (PHT) and to compare the treatment continuation rate between patients receiving conventional therapy and patients receiving individualized therapy based on genotyping of the CYP2C9*3, CYP2C19*2, and CYP2C19*3 alleles. The operational definition for the target dose of PHT used in this study was the dose that yielded a steady-state PHT concentration within the range of 15-20 mcg/mL without dose-related adverse effects. METHODS: We investigated 394 samples from 170 Japanese pediatric patients aged 9 months to 15 years to identify factors that influenced the target dose of PHT. We also analyzed the clinical records of 156 patients who commenced PHT therapy at our hospital and retrospectively assessed the time to treatment failure within 1 year after starting PHT therapy. During the study period, 17 patients underwent genotyping at the start of PHT therapy. If the patients had the CYP2C9*3, CYP2C19*2, or CYP2C19*3 alleles, the initial dose of PHT was reduced by 10%-50% according to previous reports. The other 139 patients received conventional PHT therapy. RESULTS: According to multiple regression analysis, the body weight, concomitant use of sulthiame, and the CYP2C9*3, CYP2C19*2, and CYP2C19*3 alleles influenced the target dose of PHT. Our model explained 74% of the interindividual variability of the target dose of PHT. The total withdrawal rate in the individualized therapy group and the conventional therapy group was 23.5% and 33.1%, respectively. The adjusted hazard ratio for withdrawal of PHT therapy in the individualized therapy group was 0.37 (95% confidence interval; 0.12-1.10, P = 0.074). CONCLUSIONS: These findings suggest that genotyping can help to estimate the optimum target dose of PHT and may contribute to avoid intoxication and concentration-dependent adverse effects.

[Antibodies to the glutamate receptor].

Glutamate receptors (GluRs) are classified into metabotropic GluRs and ionotropic GluRs. Ionotropic GluRs include the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) -type, kainate (KA)-type, and N-methyl-D-aspartate (NMDA)-type GluRs (NRs). Antibodies to the NRs have been detected using immunoblot, cell-based assays, and enzyme linked immunosorbent assay (ELISA). In patient with non-paraneoplastic, non-herpetic acute limbic encephalitis (NHALE), antibodies against GluN2B (GluRε2) and GluN1 (Gluζ1) are detected in the sera and CSF. In addition to the antibodies to NRs detected by ELISA, antibodies to an NR-complex detected by cell-based assay are found, not only in CSF from NHALE, but also in CSF from epilepsy, Creutzfeldt-Jakob disease (CJD), etc. Antibodies to NRs internalize mainly extra-synaptic NRs, and dissociate the connection between GluN2A and Ephrin-B2 receptor at the synapse. IgG fractions containing antibodies to NRs decrease apoptosis of cultured neurons. Antibodies to AMPA-type GluRs have been detected by immunoblot, cell-based assay and ELISA. In Rasmussen syndrome, antibodies against GluA3 (GluR3) were found to be the primary pathological factor. However the antibodies did not cause Rasmussen syndrome in mice models. These antibodies have been shown to cause excitotoxicity through GluA3, complement-dependent cytotoxicity, etc. Antibodies to GluA1/GluA2 in paraneoplastic limbic encephalitis modulate expression and localization of GluA1/GluA2 at the synapse. Antibodies to metabotropic GluRs have been detected using cell-based assays in patients with Hodgkin's lymphoma. Passive transfer of the IgG fraction from patients having antibodies to metabotropic GluR1 causes ataxia in mice.