Sumimasa Yamashita

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.

Mutations in KLHL40 Are a Frequent Cause of Severe Autosomal-Recessive Nemaline Myopathy

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

Clinical spectrum of early onset epileptic encephalopathies caused by KCNQ2 mutation.

KCNQ2 mutations have been found in patients with benign familial neonatal seizures, myokymia, or early onset epileptic encephalopathy (EOEE). In this study, we aimed to delineate the clinical spectrum of EOEE associated with KCNQ2 mutation.

Genotype–phenotype correlations in alternating hemiplegia of childhood

Objective: Clinical severity of alternating hemiplegia of childhood (AHC) is extremely variable. To investigate genotype–phenotype correlations in AHC, we analyzed the clinical information and ATP1A3 mutations in patients with AHC. Methods: Thirty-five Japanese patients who were clinically diagnosed with AHC participated in this study. ATP1A3 mutations were analyzed using Sanger sequencing. Detailed clinical information was collected from family members of patients with AHC and clinicians responsible for their care. Results: Gene analysis revealed 33 patients with de novo heterozygous missense mutations of ATP1A3: Glu815Lys in 12 cases (36%), Asp801Asn in 10 cases (30%), and other missense mutations in 11 cases. Clinical information was compared among the Glu815Lys, Asp801Asn, and other mutation groups. Statistical analysis revealed significant differences in the history of neonatal onset, gross motor level, status epilepticus, and respiratory paralysis in the Glu815Lys group compared with the other groups. In addition, 8 patients who did not receive flunarizine had severe motor deteriorations. Conclusions: The Glu815Lys genotype appears to be associated with the most severe AHC phenotype. Although AHC is not generally seen as a progressive disorder, it should be considered a disorder that deteriorates abruptly or in a stepwise fashion, particularly in patients with the Glu815Lys mutation.

Identification of ATP1A3 mutations by exome sequencing as the cause of alternating hemiplegia of childhood in Japanese patients.

Background Alternating hemiplegia of childhood (AHC) is a rare disorder characterized by transient repeated attacks of paresis and cognitive impairment. Recent studies from the U.S. and Europe have described ATP1A3 mutations in AHC. However, the genotype-phenotype relationship remains unclear. The purpose of this study was to identify the genetic abnormality in a Japanese cohort of AHC using exome analysis. Principal Findings A total of 712,558 genetic single nucleotide variations in 8 patients with sporadic AHC were found. After a series of exclusions, mutations of three genes were regarded as candidate causes of AHC. Each patient harbored a heterozygous missense mutation of ATP1A3, which included G755C, E815K, C927Y and D801N. All mutations were at highly conserved amino acid residues and deduced to affect ATPase activity of the corresponding ATP pump, the product of ATP1A3. They were de novo mutations and not identified in 96 healthy volunteers. Using Sanger sequencing, E815K was found in two other sporadic cases of AHC. In this study, E815K was found in 5 of 10 patients (50%), a prevalence higher than that reported in two recent studies [19 of 82 (23%) and 7 of 24 (29%)]. Furthermore, the clinical data of the affected individuals indicated that E815K resulted in a severer phenotype compared with other ATP1A3 mutations. Interpretation Heterozygous de novo mutations of ATP1A3 were identified in all Japanese patients with AHC examined in this study, confirming that ATP1A3 mutation is the cause of AHC.

Dominant mutations in ORAI1 cause tubular aggregate myopathy with hypocalcemia via constitutive activation of store-operated Ca2+ channels

The store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel is activated by diminished luminal Ca(2+) levels in the endoplasmic reticulum and sarcoplasmic reticulum (SR), and constitutes one of the major Ca(2+) entry pathways in various tissues. Tubular aggregates (TAs) are abnormal structures in the skeletal muscle, and although their mechanism of formation has not been clarified, altered Ca(2+) homeostasis related to a disordered SR is suggested to be one of the main contributing factors. TA myopathy is a hereditary muscle disorder that is pathologically characterized by the presence of TAs. Recently, dominant mutations in the STIM1 gene, encoding a Ca(2+) sensor that controls CRAC channels, have been identified to cause tubular aggregate myopathy (TAM). Here, we identified heterozygous missense mutations in the ORAI1 gene, encoding the CRAC channel itself, in three families affected by dominantly inherited TAM with hypocalcemia. Skeletal myotubes from an affected individual and HEK293 cells expressing mutated ORAI1 proteins displayed spontaneous extracellular Ca(2+) entry into cells without diminishment of luminal Ca(2+) or the association with STIM1. Our results indicate that STIM1-independent activation of CRAC channels induced by dominant mutations in ORAI1 cause altered Ca(2+) homeostasis, resulting in TAM with hypocalcemia.

Neuronal and glial accumulation of α- and β-synucleins in human lipidoses

A number of the lysosomal storage diseases that have now been characterized are associated with intra-lysosomal accumulation of lipids, caused by defective lysosomal enzymes. We have previously reported neuronal accumulation of both α- and β-synucleins in brain tissue of a GM2 gangliosidosis mouse model. Although α-synuclein has been implicated in several neurodegenerative disorders including Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, its functions remain largely unclear. In our present study, we have examined a cohort of human lipidosis cases, including Sandhoff disease, Tay–Sachs disease, metachromatic leukodystrophy, β-galactosialidosis and adrenoleukodystrophy, for the expression of α- and β-synucleins and the associated lipid storage levels. The accumulation of α-synuclein was found in brain tissue in not only cases of lysosomal storage diseases, but also in instances of adrenoleukodystrophy, which is a peroxisomal disease. α-synuclein was detected in both neurons and glial cells of patients with these two disorders, although its distribution was found to be disease-dependent. In addition, α-synuclein-positive neurons were also found to be NeuN-positive, whereas NeuN-negative neurons did not show any accumulation of this protein. By comparison, the accumulation of β-synuclein was detectable only in the pons of Sandhoff disease cases. This differential accumulation of α- and β-synucleins in human lipidoses may be related to functional differences between these two proteins. In addition, the accumulation of α-synuclein may also be a condition that is common to lysosomal storage diseases and adrenoleukodystrophies that show an enhanced expression of this protein upon the elevation of stored lipids.

Patients with a sodium channel alpha 1 gene mutation show wide phenotypic variation

We investigated the roles of mutations in voltage-gated sodium channel alpha 1 subunit gene (SCN1A) in epilepsies and psychiatric disorders. The SCN1A gene was screened for mutations in three unrelated Japanese families with generalized epilepsy with febrile seizure plus (GEFS+), febrile seizure with myoclonic seizures, or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC). In the family with GEFS+, one individual was affected with panic disorder and seizures, and another individual was diagnosed with Asperger syndrome and seizures. The novel mutation V1366I was found in all probands and patients with psychiatric disorders of the three families. These results suggest that SCN1A mutations may confer susceptibility to psychiatric disorders in addition to variable epileptic seizures. Unidentified modifiers may play critical roles in determining the ultimate phenotype of patients with sodium channel mutations.

A novel homozygous mutation of DARS2 may cause a severe LBSL variant.

To the Editor : Leukoencephalopathy with brain stem and spinal cord involvement, and lactate elevation (LBSL, MIM #611105) is an autosomal recessive disorder with an early childhood-to-adolescence onset. In 2003, van der Knaap et al. originally described LBSL, which is characterized by slowly progressive pyramidal, cerebellar, and dorsal column dysfunction with increased white matter lactate levels in magnetic resonance (MR) spectroscopy (1). Since the first discovery that LBSL is caused by mutations of the DARS2 gene-encoding mitochondrial aspartyl-tRNA synthetase (MtAspRS) (2), DARS2 mutations have been found in all the patients described (2–5), but none of them showed a homozygous mutation (all are compound heterozygotes), suggesting that the activity of mutant MtAspRS homodimers may be incompatible with human life (2, 5). Here, we describe, for the first time, a consanguineous family with a homozygous DARS2 mutation.

Finger cold-induced vasodilatation, sympathetic skin response, and R-R interval variation in patients with progressive spinal muscular atrophy.

To elucidate autonomic function in spinal muscular atrophy, we evaluated finger cold-induced vasodilatation, sympathetic skin response, and R—R interval variation in 10 patients with spinal muscular atrophy: 7 of type 1, 2 of type 2, and 1 of type 3. Results of finger cold-induced vasodilatation, sympathetic skin response, and R—R interval variation were compared with those of healthy children. Finger cold-induced vasodilatation was abnormal in 6 of 10patients with spinal muscular atrophy; it was normal in the healthy children. The mean sympathetic skin response latency and amplitude did not differ significantly from those of the healthy children. Amplitudes of sympathetic skin response to sound stimulation were absent or low in all six patients with spinal muscular atrophy. No significant difference was found in the mean R—R interval variation of patients with spinal muscular atrophy and healthy children. Results show that some patients with spinal muscular atrophy have autonomic dysfunction, especially sympathetic nerve hyperactivity, that resembles dysfunction observed in amyotrophic lateral sclerosis. (J Child Neurol 2005;20:871—875).