Eiji Nakagawa

Enhancement of Progenitor Cell Division in the Dentate Gyrus Triggered by Initial Limbic Seizures in Rat Models of Epilepsy

Summary: Purpose: Mitogenic effects of seizures on granule cell progenitors in the dentate gyrus were studied in two rat models of epilepsy. We investigated which stage of epileptogenesis is critical for eliciting progenitor cell division and whether seizure‐induced neuronal degeneration is responsible for the enhancement of progenitor cell division.

Clinical spectrum of SCN2A mutations expanding to Ohtahara syndrome

Objective: We aimed to investigate the possible association between SCN2A mutations and early-onset epileptic encephalopathies (EOEEs). Methods: We recruited a total of 328 patients with EOEE, including 67 patients with Ohtahara syndrome (OS) and 150 with West syndrome. SCN2A mutations were examined using high resolution melt analysis or whole exome sequencing. Results: We found 14 novel SCN2A missense mutations in 15 patients: 9 of 67 OS cases (13.4%), 1 of 150 West syndrome cases (0.67%), and 5 of 111 with unclassified EOEEs (4.5%). Twelve of the 14 mutations were confirmed as de novo, and all mutations were absent in 212 control exomes. A de novo mosaic mutation (c.3976G>C) with a mutant allele frequency of 18% was detected in one patient. One mutation (c.634A>G) was found in transcript variant 3, which is a neonatal isoform. All 9 mutations in patients with OS were located in linker regions between 2 transmembrane segments. In 7 of the 9 patients with OS, EEG findings transitioned from suppression-burst pattern to hypsarrhythmia. All 15 of the patients with novel SCN2A missense mutations had intractable seizures; 3 of them were seizure-free at the last medical examination. All patients showed severe developmental delay. Conclusions: Our study confirmed that SCN2A mutations are an important genetic cause of OS. Given the wide clinical spectrum associated with SCN2A mutations, genetic testing for SCN2A should be considered for children with different epileptic conditions.

Missense mutation of the sodium channel gene SCN2A causes Dravet syndrome

Mutations of the gene encoding the alpha2 subunit of the neuronal sodium channel, SCN2A, have been found in benign familial neonatal-infantile seizures (BFNIS). In Dravet syndrome, only one nonsense mutation of SCN2A was identified, while hundreds of mutations were found in the paralogue gene, SCN1A, which encodes the alpha1 subunit. This study examines whether SCN2A mutations are associated with Dravet syndrome. We screened for mutations of SCN1A, SCN2A and GABRG2 (the gene encoding gamma2 subunit of the GABA(A) receptor) in 59 patients with Dravet syndrome and found 29 SCN1A mutations and three missense SCN2A mutations. Among the three, one de novo SCN2A mutation (c.3935G>C: R1312T) identified in a patient was thought to affect an arginine residue in a voltage sensor of the channel and hence, to be pathogenic. This finding suggests that both nonsense mutations and missense SCN2A mutations cause Dravet syndrome.

Targeted capture and sequencing for detection of mutations causing early onset epileptic encephalopathy.

Early onset epileptic encephalopathies (EOEEs) are heterogeneous epileptic disorders caused by various abnormalities in causative genes including point mutations and copy number variations (CNVs). In this study, we performed targeted capture and sequencing of a subset of genes to detect point mutations and CNVs simultaneously.

Neocortical Layer Formation of Human Developing Brains and Lissencephalies: Consideration of Layer-Specific Marker Expression

To investigate layer-specific molecule expression in human developing neocortices, we performed immunohistochemistry of the layer-specific markers (TBR1, FOXP1, SATB2, OTX1, CUTL1, and CTIP2), using frontal neocortices of the dorsolateral precentral gyri of 16 normal controls, aged 19 gestational weeks to 1 year old, lissencephalies of 3 Miller-Dieker syndrome (MDS) cases, 2 X-linked lissencephaly with abnormal genitalia (XLAG) cases, and 4 Fukuyama-type congenital muscular dystrophy (FCMD) cases. In the fetal period, we observed SATB2+ cells in layers II-IV, CUTL1+ cells in layers II-V, FOXP1+ cells in layer V, OTX1+ cells in layers II or V, and CTIP2+ and TBR1+ cells in layers V and VI. SATB2+ and CUTL1+ cells appeared until 3 months of age, but the other markers disappeared after birth. Neocortices of MDS and XLAG infants revealed SATB2+, CUTL1+, FOXP1+, and TBR1+ cells diffusely located in the upper layers. In fetal FCMD neocortex, neurons labeled with the layer-specific markers located over the glia limitans. The present study provided new knowledge indicating that the expression pattern of these markers in the developing human neocortex was similar to those in mice. Various lissencephalies revealed abnormal layer formation by random migration.

Copy-number variations on the X chromosome in Japanese patients with mental retardation detected by array-based comparative genomic hybridization analysis

X-linked mental retardation (XLMR) is a common, clinically complex and genetically heterogeneous disease arising from many mutations along the X chromosome. Although research during the past decade has identified >90 XLMR genes, many more remain uncharacterized. In this study, copy-number variations (CNVs) were screened in individuals with MR from 144 families by array-based comparative genomic hybridization (aCGH) using a bacterial artificial chromosome-based X-tiling array. Candidate pathogenic CNVs (pCNVs) were detected in 10 families (6.9%). Five of the families had pCNVs involving known XLMR genes, duplication of Xq28 containing MECP2 in three families, duplication of Xp11.22-p11.23 containing FTSJ1 and PQBP1 in one family, and deletion of Xp11.22 bearing SHROOM4 in one family. New candidate pCNVs were detected in five families as follows: identical complex pCNVs involved in dup(X)(p22.2) and dup(X)(p21.3) containing part of REPS2, NHS and IL1RAPL1 in two unrelated families, duplication of Xp22.2 including part of FRMPD4, duplication of Xq21.1 including HDX and deletion of Xq24 noncoding region in one family, respectively. Both parents and only mother samples were available in six and three families, respectively, and pCNVs were inherited from each of their mothers in those families other than a family of the proband with deletion of SHROOM4. This study should help to identify the novel XLMR genes and mechanisms leading to MR and reveal the clinical conditions and genomic background of XLMR.

Inflammatory changes in infantile-onset LMNA-associated myopathy.

Mutations in LMNA cause wide variety of disorders including Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, and congenital muscular dystrophy. We recently found a LMNA mutation in a patient who was previously diagnosed as infantile onset inflammatory myopathy. In this study, we screened for LMNA mutations in 20 patients suspected to have inflammatory myopathy with onset at 2years or younger. The diagnosis of inflammatory myopathy was based on muscle pathology with presence of perivascular cuffing and/or endomysial/perimysial lymphocyte infiltration. We identified heterozygous LMNA mutations in 11 patients (55%), who eventually developed joint contractures and/or cardiac involvement after the infantile period. Our findings suggest that LMNA mutation should be considered in myopathy patients with inflammatory changes during infancy, and that this may help avoid life-threatening events associated with laminopathy.

Neurobehavioral and hemodynamic evaluation of Stroop and reverse Stroop interference in children with attention-deficit/hyperactivity disorder

Failure of executive function (EF) is a core symptom of attention-deficit/hyperactivity disorder (ADHD). However, various results have been reported and sufficient evidence is lacking. In the present study, we evaluated the characteristics of children with ADHD using the Stroop task (ST) and reverse Stroop task (RST) that reflects the inhibition function of EF. We compared children with ADHD, typically developing children (TDC), and children with autism spectrum disorder (ASD), which is more difficult to discriminate from ADHD. A total of 10 children diagnosed with ADHD, 15 TDC, and 11 children diagnosed with ASD, all matched by age, sex, language ability, and intelligence quotient, participated in this study. While each subject performed computer-based ST and RST with a touch panel, changes in oxygenated hemoglobin (oxy-Hb) were measured in the prefrontal cortex (PFC) by near-infrared spectroscopy (NIRS) to correlate test performance with neural activity. Behavioral performance significantly differed among 3 groups during RST but not during ST. The ADHD group showed greater color interference than the TDC group. In addition, there was a negative correlation between right lateral PFC (LPFC) activity and the severity of attention deficit. Children with ADHD exhibit several problems associated with inhibition of color, and this symptom is affected by low activities of the right LPFC. In addition, it is suggested that low hemodynamic activities in this area are correlated with ADHD.

TULIP1 (RALGAPA1) haploinsufficiency with brain development delay

A novel microdeletion of 14q13.1q13.3 was identified in a patient with developmental delay and intractable epilepsy. The 2.2-Mb deletion included 15 genes, of which TULIP1 (approved gene symbol: RALGAPA1)was the only gene highly expressed in the brain. Western blotting revealed reduced amount of TULIP1 in cell lysates derived from immortalized lymphocytes of the patient, suggesting the association between TULIP1 haploinsufficiency and the patients phenotype, then 140 patients were screened for TULIP1 mutations and four missense mutations were identified. Although all four missense mutations were common with parents, reduced TULIP1 was observed in the cell lysates with a P297T mutation identified in a conserved region among species. A full-length homolog of human TULIP1 was identified in zebrafish with 72% identity to human. Tulip1 was highly expressed in zebrafish brain, and knockdown of which resulted in brain developmental delay. Therefore, we suggest that TULIP1 is a candidate gene for developmental delay.

Decreased resting energy expenditure in patients with Duchenne muscular dystrophy.

BACKGROUND Skeletal muscle metabolism is a major determinant of resting energy expenditure (REE). Although the severe muscle loss that characterizes Duchenne muscular dystrophy (DMD) may alter REE, this has not been extensively investigated. METHODS We studied REE in 77 patients with DMD ranging in age from 10 to 37 years using a portable indirect calorimeter, together with several clinical parameters (age, height, body weight (BW), body mass index (BMI), vital capacity (VC), creatine kinase, creatinine, albumin, cholinesterase, prealbumin), and assessed their influence on REE. In addition, in 12 patients maintaining a stable body weight, the ratio of energy intake to REE was calculated and defined as an alternative index for the physical activity level (aPAL). RESULTS REE (kcal/day, mean±SD) in DMD patients was 1123 (10-11 years), 1186±188 (12-14 years), 1146±214 (15-17 years), 1006±136 (18-29 years) and 1023±97 (≥30 years), each of these values being significantly lower than the corresponding control (p<0.0001). VC (p<0.001) was the parameter most strongly associated with REE, followed by BMI (p<0.01) and BW (p<0.05). The calculated aPAL values were 1.61 (10-11 years), 1.19 (12-14 years), 1.16 (15-17 years), and 1.57 (18-29 years). CONCLUSION The REE in DMD patients was significantly lower than the normal value in every age group, and strongly associated with VC. Both the low REE and PAL values during the early teens, resulting in a low energy requirement, might be related to the obesity that frequently occurs in this age group. In contrast, the high PAL value in the late stage of the disease, possibly due to the presence of respiratory failure, may lead to a high energy requirement, and thus become one of the risk factors for development of malnutrition.