Tojo Nakayama

A homozygous mutation of voltage‐gated sodium channel βI gene SCN1B in a patient with Dravet syndrome

Dravet syndrome is a severe form of epileptic encephalopathy characterized by early onset epileptic seizures followed by ataxia and cognitive decline. Approximately 80% of patients with Dravet syndrome have been associated with heterozygous mutations in SCN1A gene encoding voltage‐gated sodium channel (VGSC) αI subunit, whereas a homozygous mutation (p.Arg125Cys) of SCN1B gene encoding VGSC βI subunit was recently described in a patient with Dravet syndrome. To further examine the involvement of homozygous SCN1B mutations in the etiology of Dravet syndrome, we performed mutational analyses on SCN1B in 286 patients with epileptic disorders, including 67 patients with Dravet syndrome who have been negative for SCN1A and SCN2A mutations. In the cohort, we found one additional homozygous mutation (p.Ile106Phe) in a patient with Dravet syndrome. The identified homozygous SCN1B mutations indicate that SCN1B is an etiologic candidate underlying Dravet syndrome.

Deletions of SCN1A 5′ genomic region with promoter activity in Dravet syndrome

Mutations involving the voltage‐gated sodium channel αI gene SCN1A are major genetic causes of childhood epileptic disorders, as typified by Dravet syndrome. Here we investigated the upstream regions of the SCN1A 5′ noncoding exons and found two major regions with promoter activity. These two major promoters were simultaneously active in various brain regions and in most neurons. Using multiplex ligation‐dependent probe amplification (MLPA) assays with probes for the 5′ noncoding exons, their upstream regions, and all coding exons of SCN1A, we investigated 130 epileptic patients who did not show any SCN1A mutations by sequence analysis of all coding exons and exon–intron boundaries. Among 71 Dravet syndrome patients, we found two patients with heterozygous microdeletions removing the 5′ noncoding exons and regions with promoter activity but not affecting the coding exons. We also identified four patients with deletions/duplication in the coding region. One patient with symptomatic focal epilepsy also showed a deletion in the coding region. This study provides the first case of microdeletion limited to the SCN1A 5′ promoter region with the coding sequence preserved, and indicates the critical involvement of this upstream region in the molecular pathology of Dravet syndrome. Hum Mutat 31:–11, 2010.

Complete remission of seizures after corpus callosotomy

OBJECT Corpus callosotomy is usually intended to alleviate-not to achieve total control of-epileptic seizures. A few patients experience complete seizure control after callosotomy, but the associated clinical factors are unknown. The object of this study was to investigate clinical factors associated with long-term seizure remission after total corpus callosotomy in patients with infantile or early childhood onset epilepsy. METHODS Thirteen consecutive patients with infantile or early childhood onset epilepsy underwent 1-stage total corpus callosotomy for alleviation of seizures. Their age at surgery ranged from 1 year and 5 months to 24 years (median 7 years). Eleven patients had West syndrome at the onset of disease, and the other 2 had Lennox-Gastaut syndrome. All patients suffered from spasms, axial tonic seizures, or atonic seizures. Six patients had proven etiology of epilepsy, including tuberous sclerosis, polymicrogyria, trauma, and Smith-Magenis syndrome. The association between postoperative seizure freedom and preoperative factors including age at surgery, no MRI abnormalities, proven etiology, and focal electroencephalographic epileptiform discharges was examined. RESULTS Postoperative seizure freedom was achieved in 4 of 13 patients for a minimum of 12 months. All 4 patients had no MRI abnormalities and no identified etiology. None of the 8 patients with MRI abnormality, 6 patients with known etiology of epilepsy, or 4 patients aged older than 10 years at surgery achieved seizure freedom. Two of the 7 patients with focal electroencephalographic abnormalities became seizure free. Absence of MRI abnormalities was significantly associated with postoperative seizure freedom (p < 0.01). CONCLUSIONS Complete seizure remission is achieved after total corpus callosotomy in a subgroup of patients with intractable epilepsy following West syndrome or Lennox-Gastaut syndrome. One-stage total corpus callosotomy at a young age may provide a higher rate of seizure freedom, especially for patients with no MRI abnormalities and no identified etiology of epilepsy.

Mutations in PYCR2, Encoding Pyrroline-5-Carboxylate Reductase 2, Cause Microcephaly and Hypomyelination

Despite recent advances in understanding the genetic bases of microcephaly, a large number of cases of microcephaly remain unexplained, suggesting that many microcephaly syndromes and associated genes have yet to be identified. Here, we report mutations in PYCR2, which encodes an enzyme in the proline biosynthesis pathway, as the cause of a unique syndrome characterized by postnatal microcephaly, hypomyelination, and reduced cerebral white-matter volume. Linkage mapping and whole-exome sequencing identified homozygous mutations (c.355C>T [p.Arg119Cys] and c.751C>T [p.Arg251Cys]) in PYCR2 in the affected individuals of two consanguineous families. A lymphoblastoid cell line from one affected individual showed a strong reduction in the amount of PYCR2. When mutant cDNAs were transfected into HEK293FT cells, both variant proteins retained normal mitochondrial localization but had lower amounts than the wild-type protein, suggesting that the variant proteins were less stable. A PYCR2-deficient HEK293FT cell line generated by genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system showed that PYCR2 loss of function led to decreased mitochondrial membrane potential and increased susceptibility to apoptosis under oxidative stress. Morpholino-based knockdown of a zebrafish PYCR2 ortholog, pycr1b, recapitulated the human microcephaly phenotype, which was rescued by wild-type human PYCR2 mRNA, but not by mutant mRNAs, further supporting the pathogenicity of the identified variants. Hypomyelination and the absence of lax, wrinkly skin distinguishes this condition from that caused by previously reported mutations in the gene encoding PYCR2s isozyme, PYCR1, suggesting a unique and indispensable role for PYCR2 in the human CNS during development.

Mutations in mitochondrial enzyme GPT2 cause metabolic dysfunction and neurological disease with developmental and progressive features

Significance We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in a neurological syndrome involving intellectual disability, reduced brain growth, and progressive motor symptoms. We show that the mutations inactivate the enzyme. GPT2 catalyzes the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. The GPT2 gene demonstrates expression in brain postnatally, and the protein localizes to mitochondria. As in humans, Gpt2-null mice exhibit reduced brain growth. Furthermore, mutant mouse brains show abnormal metabolite levels, including in pathways involving amino acid metabolism, the TCA cycle, and neuroprotective mechanisms. Our study identifies GPT2 as an important mitochondrial enzyme in disease that has general relevance to developmental and potentially to neurodegenerative mechanisms. Mutations that cause neurological phenotypes are highly informative with regard to mechanisms governing human brain function and disease. We report autosomal recessive mutations in the enzyme glutamate pyruvate transaminase 2 (GPT2) in large kindreds initially ascertained for intellectual and developmental disability (IDD). GPT2 [also known as alanine transaminase 2 (ALT2)] is one of two related transaminases that catalyze the reversible addition of an amino group from glutamate to pyruvate, yielding alanine and α-ketoglutarate. In addition to IDD, all affected individuals show postnatal microcephaly and ∼80% of those followed over time show progressive motor symptoms, a spastic paraplegia. Homozygous nonsense p.Arg404* and missense p.Pro272Leu mutations are shown biochemically to be loss of function. The GPT2 gene demonstrates increasing expression in brain in the early postnatal period, and GPT2 protein localizes to mitochondria. Akin to the human phenotype, Gpt2-null mice exhibit reduced brain growth. Through metabolomics and direct isotope tracing experiments, we find a number of metabolic abnormalities associated with loss of Gpt2. These include defects in amino acid metabolism such as low alanine levels and elevated essential amino acids. Also, we find defects in anaplerosis, the metabolic process involved in replenishing TCA cycle intermediates. Finally, mutant brains demonstrate misregulated metabolites in pathways implicated in neuroprotective mechanisms previously associated with neurodegenerative disorders. Overall, our data reveal an important role for the GPT2 enzyme in mitochondrial metabolism with relevance to developmental as well as potentially to neurodegenerative mechanisms.

Repeat magnetoencephalography and surgeries to eliminate atonic seizures of non-lesional frontal lobe epilepsy.

A 7-year-old boy presented with intractable head nodding and atonic seizures since the age of 3 years and severe behavior problems unsuitable for video EEG monitoring. We performed magnetoencephalography (MEG) 4 times: before (1) corpus callosotomy, (2) left frontal cortical resection with multiple subpial transection and (3) resection of residual MEG spike sources, which eliminated seizures. Repeat MEGs consistently localized the epileptogenic zone in a patient with intractable residual non-lesional left frontal lobe epilepsy.

Hypoperfusion in caudate nuclei in patients with brain–lung–thyroid syndrome

Mutations in NKX2-1 cause neurological, pulmonary, and thyroid hormone impairment. Recently, the disease was named brain-lung-thyroid syndrome. Here, we report three patients with brain-lung-thyroid syndrome. All patients were unable to walk until 24 months of age, and still have a staggering gait, without mental retardation. They have also had choreoathetosis since early infancy. Genetic analysis of NKX2-1 revealed a novel missense mutation (p.Val205Phe) in two patients who were cousins and their maternal families, and a novel 2.6-Mb deletion including NKX2-1 on chromosome 14 in the other patient. Congenital hypothyroidism was not detected on neonatal screening in the patient with the missense mutation, and frequent respiratory infections were observed in the patient with the deletion in NKX2-1. Oral levodopa did not improve the gait disturbance or involuntary movement. The results of (99m)Tc-ECD single-photon emission computed tomography (ECD-SPECT) analyzed using the easy Z-score imaging system showed decreased cerebral blood flow in the bilateral basal ganglia, especially in the caudate nuclei, in all three patients, but no brain magnetic resonance imaging (MRI) abnormalities. These brain nuclear image findings indicate that NKX2-1 haploinsufficiency causes dysfunction of the basal ganglia, especially the caudate nuclei, resulting in choreoathetosis and gait disturbance in this disease.

Deficient activity of alanyl-tRNA synthetase underlies an autosomal recessive syndrome of progressive microcephaly, hypomyelination, and epileptic encephalopathy

Aminoacyl‐transfer RNA (tRNA) synthetases ligate amino acids to specific tRNAs and are essential for protein synthesis. Although alanyl‐tRNA synthetase (AARS) is a synthetase implicated in a wide range of neurological disorders from Charcot‐Marie‐Tooth disease to infantile epileptic encephalopathy, there have been limited data on their pathogenesis. Here, we report loss‐of‐function mutations in AARS in two siblings with progressive microcephaly with hypomyelination, intractable epilepsy, and spasticity. Whole‐exome sequencing identified that the affected individuals were compound heterozygous for mutations in AARS gene, c.2067dupC (p.Tyr690Leufs*3) and c.2738G>A (p.Gly913Asp). A lymphoblastoid cell line developed from one of the affected individuals showed a strong reduction in AARS abundance. The mutations decrease aminoacylation efficiency by 70%–90%. The p.Tyr690Leufs*3 mutation also abolished editing activity required for hydrolyzing misacylated tRNAs, thereby increasing errors during aminoacylation. Our study has extended potential mechanisms underlying AARS‐related disorders to include destabilization of the protein, aminoacylation dysfunction, and defective editing activity.

Parental satisfaction and seizure outcome after corpus callosotomy in patients with infantile or early childhood onset epilepsy

PURPOSE To elucidate the benefit of corpus callosotmy in terms of parental satisfaction and seizure outcome. METHOD This study included 16 consecutive patients with infantile or early childhood onset epilepsy who underwent total corpus callosotomy for alleviation of seizures. Questionnaires were sent anonymously to the parents asking about relative changes in seizures and about parental satisfaction for the post-operative outcome. RESULTS The improvements in frequency, intensity, and duration of seizures were correlated with the level of satisfaction (Spearmans rank-order correlation coefficient, ρ=0.87, 0.93, and 0.75, respectively). The highest level of satisfaction was only seen in patients who achieved freedom from all seizures or drop attacks. CONCLUSION Complete seizure freedom and freedom from drop attacks are important goals of corpus callosotomy for parental satisfaction. These factors should be considered in assessing post-operative outcome after corpus callosotomy.

The usefulness of subtraction ictal SPECT and ictal near-infrared spectroscopic topography in patients with West syndrome.

The recent findings on subtraction ictal SPECT and ictal near-infrared spectroscopic topography in patients with West syndrome were summarized and its availability for presurgical evaluation was discussed. The subtraction ictal SPECT study in patients with West syndrome demonstrated the cortical epileptic region and subcortical involvement, which may consist of epilepsy networks related to the spasms. Moreover, subtraction ictal SPECT may have predictive power for short-term seizure outcome. Patients with a symmetric hyperperfusion pattern are predicted to have a better seizure outcome, whereas patients with asymmetric hyperperfusion pattern may develop poor seizure control. Importantly, asymmetric MRI findings had no predictive power for seizure outcome. Multichannel near-infrared spectroscopic topography applied to the patients with West syndrome detected an increase in regional cerebral blood volume in multiple areas which were activated either simultaneously or sequentially during spasms. Topographic changes in cerebral blood volume were closely correlated with spasm phenotype, suggesting that the cortex is involved in the generation of spasms. In conclusion, subtraction ictal SPECT may be considered as a useful tool for presurgical evaluation of patients with West syndrome and investigation of the pathophysiology of spasms. The ictal near-infrared spectroscopic topography should be more investigated to see if this is useful tool for presurgical evaluation.