Novel de novo POLR3B mutations responsible for demyelinating Charcot-Marie-Tooth disease in Japan.

BACKGROUND: Biallelic POLR3B mutations cause a rare hypomyelinating leukodystrophy. De novo POLR3B heterozygous mutations were recently associated with afferent ataxia, spasticity, variable intellectual disability, and epilepsy, and predominantly demyelinating sensorimotor peripheral neuropathy. METHODS: We performed whole-exome sequencing (WES) of DNA samples from 804 Charcot-Marie-Tooth (CMT) cases that could not be genetically diagnosed by DNA-targeted resequencing microarray using next-generation sequencers. Using WES data, we analyzed the POLR3B mutations and confirmed their clinical features. RESULTS: We identified de novo POLR3B heterozygous missense mutations in two patients. These patients presented with early-onset demyelinating sensorimotor neuropathy without ataxia, spasticity, or cognitive impairment. Patient 1 showed mild cerebellar atrophy and spinal cord atrophy on magnetic resonance imaging and eventually died of respiratory failure in her 50s. We classified these mutations as pathogenic based on segregation studies, comparison with control database, and in silico analysis. CONCLUSION: Our study is the third report on patients with demyelinating CMT harboring heterozygous POLR3B mutations and verifies the pathogenicity of POLR3B mutations in CMT. Although extremely rare in our large Japanese case series, POLR3B mutations should be added to the CMT-related gene panel for comprehensive genetic screening, particularly for patients with early-onset demyelinating CMT.

Clinical Usefulness of Serum Krebs von den Lungen-6 for Detecting Chronic Aspiration in Children with Severe Motor and Intellectual Disabilities.

OBJECTIVE: To determine whether the serum level of Krebs von den Lungen-6 (KL-6), a circulating high-molecular weight glycoprotein and a diagnostic biomarker of interstitial lung diseases, is a clinically useful biomarker for detecting chronic aspiration in children with severe motor and intellectual disabilities (SMIDS). STUDY DESIGN: Children with SMIDS undergoing videofluorography for assessment of dysphagia were prospectively evaluated. Based on the videofluorography results, the participants were classified into aspiration and non-aspiration groups. Age, sex, white blood cell count, and serum levels of C-reactive protein, lactate dehydrogenase, albumin, and KL-6 were compared between the 2 groups. Binary logistic regression was performed to identify factors independently associated with the presence of aspiration. RESULTS: A total of 66 patients participated in this study, 37 who were classified as the aspiration group and 29 as the non-aspiration group. The serum KL-6 level in the aspiration group was significantly higher than that in the non-aspiration group (median, 344 U/mL vs 207 U/mL, P < .01). Logistic regression modeling showed that the number of prescribed antiepileptic drugs (OR, 1.978; 95% CI, 1.217, 3.214; P < .01) and serum KL-6 level (OR, 1.012; 95% CI, 1.005, 1.019; P < .01) were independent predictors of aspiration. CONCLUSIONS: The study demonstrated that the KL-6 level is significantly higher in children with SMIDS who aspirate than in those who do not. KL-6 shows promise as a biomarker for chronic lung disease due to aspiration in these children.

Carnitine deficiency: Risk factors and incidence in children with epilepsy.

BACKGROUND: Carnitine deficiency is relatively common in epilepsy; risk factors reportedly include combination antiepileptic drug (AED) therapy with valproic acid (VPA), young age, intellectual disability, diet and enteral or parenteral feeding. Few studies have examined the correlation between each risk factor and carnitine deficiency in children with epilepsy. We examined the influence of these risk factors on carnitine deficiency, and identified a formula to estimate plasma free carnitine concentration in children with epilepsy. METHODS: Sixty-five children with epilepsy and 26 age-matched controls were enrolled. Plasma carnitine concentrations were measured using an enzyme cycling assay, and correlations were sought with patients' other clinical characteristics. RESULTS: Carnitine deficiency was found in approximately 17% of patients with epilepsy and was significantly associated with carnitine-free enteral formula only by tube feeding, number of AEDs taken (independent of VPA use), body weight (BW), body height and Gross Motor Function Classification System (GMFCS) score. Stepwise multiple linear regression analysis indicated that carnitine concentration (in µmol/L) could be accurately estimated from a formula that does not require blood testing: 42.44+0.14×(BW in kg)-18.16×(feeding)-3.19×(number of AEDs), where feeding was allocated a score of 1 for carnitine-free enteral formula only by tube feeding and 0 for taking food orally (R(2)=0.504, P<0.001). CONCLUSIONS: Carnitine-free enteral formula only by tube feeding, multiple AED treatment and low BW are risk factors for carnitine deficiency in children with epilepsy. l-carnitine should be administered to children at risk of deficiency to avoid complications. Treatment decisions can be informed using an estimation formula that does not require blood tests.

Postnatal interleukin-1ß enhances adulthood seizure susceptibility and neuronal cell death after prolonged experimental febrile seizures in infantile rats.

Febrile seizures (FS) are recognized as an antecedent to the development of temporal lobe epilepsy with hippocampal sclerosis (TLE-HS), but it is unclear whether prolonged FS are a direct cause of TLE-HS. Here, we used a rat model of infantile FS to study the effects of inflammatory cytokines on seizure susceptibility and neuronal death in adults. Prolonged hyperthermia-induced seizures (pHS) were induced in male Lewis rats at post natal day (P) 10. Cytokines were administered twice intranasally, once immediately after pHS and once the following day. The effects of intranasal interleukin (IL)-1ß or tumor necrosis factor (TNF) α were tested in rats undergoing a single episode of pHS (P10) and in rats undergoing repeated pHS (P10 and P12). Seizure susceptibility was tested at P70-73 by quantifying the seizure onset time (SOT) after kainic acid administration, and neuronal cell injury and gliosis in adulthood. SOT significantly reduced in rats receiving IL-1ß together with repeated pHS, whereas no significant effects were seen in rats receiving IL-1ß after a single pHS episode, or in rats receiving TNFα. Hippocampal neuronal cell loss was observed in the CA3 region of rats receiving IL-1ß together with repeated pHS; however, there was no significant change in gliosis among each group. Our results are consistent with the hypothesis that excessive production of IL-1ß after repeated prolonged FS can enhance adult seizure susceptibility and neuronal cell death, and might contribute to the development of TLE-HS.

L-arginine is effective in stroke-like episodes of MELAS associated with the G13513A mutation.

We report a case involving a 15-year-old boy with MELAS (G13513A mutation) who developed several stroke-like episodes in a short period of time. Intravenous administration of l-arginine during the acute phase of the stroke-like episodes reduced symptoms immediately, and oral supplementation of l-arginine successfully prevented further stroke-like episodes. This is the first report on effective l-arginine therapy in MELAS associated with the G13513A mutation.

Activation of central adenosine A(2A) receptors lowers the seizure threshold of hyperthermia-induced seizure in childhood rats.

Adenosine is a potent neuromodulator in the central nervous system (CNS). The functional deterioration of adenosine A(1) receptors in the CNS was reported to cause a failure of termination of seizures and to a lower seizure threshold of hyperthermia-induced seizures (HS) in childhood rats, which may contribute to adenosine-related convulsive disorders such as theophylline-associated seizures in childhood patients. In contrast to the inhibitory effect of adenosine A(1) receptors, the function of adenosine A(2A) receptors remains controversial. To clarify the function of adenosine A(2A) receptors in childhood convulsive disorders associated to hyperthermia, we investigated the in vivo interaction between adenosine A(2A) receptors and their ligands in HS in childhood rats. Adenosine selective A(2A) receptor ligands were injected intraperitoneally before HS. We measured brain temperature at the onset of seizures and the mortality rate after HS. We found that brain temperature at seizure onset was significantly higher in the A(2A) receptor antagonist group compared with that in the control group (p<0.05), and there was no significant difference in mortality among the groups. In contrast, brain temperature at seizure onset was significantly lower in the A(2A) receptor agonist group compared with that in the control group (p<0.05), and mortality was significantly higher in the A(2A) agonist group compared with that in the control group (p<0.001). The activation of the adenosine A(2A) receptor might enhance seizures associated to hyperthermia in the childhood human brain, and be involved in the pathogenesis of sudden unexpected death in epilepsy (SUDEP) in childhood patients with convulsive disorders.

Adenosine A1 receptor blockage mediates theophylline-associated seizures.

Theophylline-associated seizures (TAS) often progress to prolonged or treatment-resistant convulsions. Theophylline is a nonselective adenosine receptor antagonist. Adenosine is an endogenous anticonvulsant that can terminate seizures. Fever and young age have been reported to be risk factors for TAS. To elucidate the mechanism of TAS, we investigated the effect of theophylline and adenosine receptor ligands on hyperthermia-induced seizures in juvenile rats. The treatment dose of theophylline or control saline was injected intraperitoneally 1 h before hyperthermia-induced seizures. The seizure threshold in the theophylline group was significantly lower and seizure duration was significantly longer than those in the control group. The addition of a selective adenosine A(1) receptor agonist and an adenosine kinase inhibitor completely counteracted the effects of theophylline. Moreover, a selective A(1) antagonist caused a significantly longer seizure duration compared with the control. These findings suggest that blockage of the adenosine A(1) receptor is the main cause of TAS.

Interleukin-1beta enhances susceptibility to hyperthermia-induced seizures in developing rats.

Cytokines have been shown to influence susceptibility to febrile seizures and epilepsy. In this study, the role of interleukin-1beta (IL-1beta) was examined in developing rats. IL-1beta and interleukin-1 receptor antagonist (IL-1ra) were administered to developing rats, and seizures were induced by moist warm air. Twenty male Lewis rats (21-23 days old) were divided into two groups (IL-1beta and saline control groups) and two holes were made in the skull for EEG electrodes. We applied human recombinant IL-1beta intra-nasally 1h before seizures induced by moist warm air. The brain temperature at the appearance of seizure discharges on EEG, and the latency time from the hyperthermia onset until the appearance of seizure discharges on EEG were measured. And the same study using IL-1ra was performed. The median brain temperature for the IL-1beta group, 42.6 degrees C (range: 41.8-43.0), was significantly lower than that for the control, 42.9 (42.3-43.4) (P=0.043). The brain temperature for the IL-1ra group, 43.3 (42.8-43.7), was significantly higher than that for the control, 42.9 (42.2-43.5) (P=0.011), and the latency time for the IL-1ra group, 398s (270-561), was significantly longer than that for the control, 325 (252-462) (P=0.035). These results demonstrate that IL-1beta promotes hyperthermia-induced seizures in developing rats.

[Dysphagia and videofluoroscopic examination of swallowing in patients with severe motor and intellectual disabilities, and the results of dysphagia rehabilitation for these patients].

A dysphagia rehabilitation team at National Ehime Hospital treated fifty-seven patients with severe motor and intellectual disabilities suspected of having dysphagia. The rehabilitation team graded the degree of dysphagia by observing the patients at mealtimes. The grades were compared with the videofluoroscopic examination of swallowing results. There was some correlation with the dysphagia grades and videofluoroscopic examination of swallowing results (solid food:p = - 0.434, n =54, p < 0.0001, liquid:p = - 0.482, n = 54, p = 0.005). There were cases in which manifestations of severe dysphagia such as retention of solid food in the pharynx could only be detected on videofluoroscopic examination of swallowing. Dietary manipulation allowed shortening of the time taken at mealtimes for some patients. The dysphagia rehabilitation was useful in many cases in decreasing the number of days with fever, and improving the eating functional level, but the results as a whole were not statistically significant.

Interleukin-6 attenuates hyperthermia-induced seizures in developing rats.

Previous studies indicated that several cytokines influenced the seizure propensity in convulsive disorders and were the cause of encephalopathies in childhood. We studied the role of one inflammatory cytokine, interleukin-6 (IL-6), in hyperthermia-induced seizures in developing rats. Twenty-four male Lewis rats (23-28 days old) were divided into three groups (n=8/IL-6 (500 ng), IL-6 (50 ng), and saline control groups). We applied human recombinant IL-6 intra-nasally to developing rats 1h before seizures induced by moist heated air (50 degrees C). The seizure latency was defined as the time from hyperthermia onset until the appearance of continuous seizure discharges on electroencephalography (EEG), and the seizure duration as the duration of continuous spike and wave discharges on EEG. Five of the eight rats in the IL-6 (500 ng) group, two in the IL-6 (50 ng) group, and one in the control group exhibited no seizure discharges during the 360 s heating period. In these cases, the seizure latency time was regarded as 360 s and the seizure duration time as 0 s. The median seizure latency for the IL-6 (500 ng) group, 360 s (range: 256-360), was significantly longer than that for the control one, 249 (121-360) (P<0.05). The seizure duration for the IL-6 (500 ng) group, 0 s (0-20), was significantly shorter than that for the control one, 33 (0-76) (P<0.025). Also, the adenosine receptor antagonist, aminophylline, prevented these effects of IL-6 on hyperthermia-induced seizures. These results indicate that IL-6 plays an anti-convulsive role through the adenosine system in hyperthermia-induced seizures, which might be relevant as to human febrile seizures.

Successful botulinum toxin treatment of dysphagia in a spinal muscular atrophy type 2 patient.

Prominent dysphagia is seen among patients with spinal muscular atrophy (SMA) type 2, especially at the late stage of their disease progression. Nasogastric tube feeding and gastrostomy are commonly utilized to maintain their nutritional status. However, choosing a treatment strategy to maintain appropriate nutritional status is often complicated by multiple factors, such as physical conditions and social aspects. We report a 21-year-old man with SMA type 2 who has been suffering from severe dysphagia. The findings at video-fluoroscopic swallow study (VSS) were consistent with a diagnosis of cricopharyngeal dysphagia. His dysphagia was successfully treated with percutaneous injection of botulinum toxin A (BTA) into the cricopharyngeal muscle. Our result demonstrates that administration of BTA is one of the effective treatment choices for dysphagia in SMA patients.

Clinical investigation of genetic contributions to childhood-onset epilepsies and epileptic syndromes.

Although there have been major advances in the understanding of the molecular bases of certain inherited epilepsy syndromes, clinical studies are still needed to verify the possible genetic contributions to common epilepsies. We examined the proportions of positive family histories of epilepsy (within second-degree relatives) and consanguinity (within first-degree relatives) in 311 probands with childhood-onset epilepsy, and found that they had high family history rates of epilepsy (19.3%) and consanguinity (6.1%). A positive family history of epilepsy was found more in probands with generalized epilepsy than in ones with localization-related epilepsy, and more in probands with idiopathic/cryptogenic epilepsy than in ones with symptomatic epilepsy. However, on analysis after the symptomatic epilepsies had been divided into two categories, probands with pre- or perinatal symptomatic generalized epilepsy and ones with postnatal symptomatic localization-related epilepsy showed high positive family history rates, similar to ones with idiopathic/cryptogenic epilepsy. On the other hand, a positive family history of consanguinity was noted more in probands with generalized epilepsy than in ones with localization-related epilepsy, but there was no significant difference between probands with idiopathic/cryptogenic epilepsy and ones with symptomatic epilepsy. These findings suggest that in addition to the hereditary effect on idiopathic/cryptogenic epilepsy, a genetic susceptibility may contribute to the development of pre- or perinatal symptomatic generalized epilepsy, and to that of postnatal symptomatic localization-related epilepsy. Furthermore, a genetic predisposition seems to have an influence through consanguinity on the etiologies of both idiopathic/cryptogenic and symptomatic generalized epilepsies.

Sequential changes of brain CT and MRI after febrile status epilepticus in a 6-year-old girl.

Brain CT or MRI occasionally shows transient or permanent changes in the brain after status epilepticus (SE). The mechanism for these changes has not been well elucidated. We performed repeated imaging studies on a patient with febrile SE characterized by right hemiconvulsion. CT showed transient mild edema on both hemispheres immediately after the cessation of SE. The edema improved the next day. But aphasia and right hemiparesis were observed. On day 17, CT revealed edema on left hemisphere and MRI showed a high signal intensity in cortex and subcortical white matter of the left hemisphere on T2-weighted images. Although right hemiparesis and aphasia were improved, severe atrophy of the left hemisphere was noted on CT and MRI. The results suggest that brain edema observed in several days after SE but not edema observed immediately after the cessation of SE is more pathological for the permanent brain damage. Possible mechanisms of the initial brain edema and the second edema preceded severe atrophy in left hemisphere were discussed.