The clinical spectrum associated with ATP1A2 variants in Chinese pediatric patients

PurposeTo evaluate the clinical spectrum associated with ATP1A2 variants in Chinese children with hemiplegia, migraines, encephalopathy or seizures.MethodsSixteen children (12 males and 4 females), including ten patients with ATP1A2 variants whose cases had been published previously, were identified using next-generation sequencing.ResultsFifteen patients had FHM2 (familial hemiplegic migraine type 2), including three who had AHC (alternating hemiplegia of childhood) and one who had drug-resistant focal epilepsy. Thirteen patients had DD (developmental delay). The onset of febrile seizures, which occurred between 5 months and 2 years 5 months (median 1 year 3 months) was earlier than the onset of HM (hemiplegic migraine), which occurred between 1 year 5 months and 13 years (median 3 years 11 months). Disturbance of consciousness subsided first, at 40 h to 9 days (median 4.5 days); hemiplegia and aphasia were resolved slowly, taking 30 min to 6 months (median 17.5 days) for the former and 24 h to over 1 year (median 14.5 days) for the latter. Cranial MRI showed edema in the cerebral hemispheres, mainly the left hemisphereacute attacks. All thirteen FHM2 patients recovered to baseline in 30 min to 6 months. Fifteen patients had between 1 and 7 (median 2) total attacks between the baseline and follow-up timepoints. We report twelve missense variants, including a novel variant ATP1A2 variant, p.G855E.ConclusionsThe known genotypic and phenotypic spectra of Chinese patients with ATP1A2-related disorders were further expanded. Recurrent febrile seizures and DD combined with paroxysmal hemiplegia and encephalopathy should raise the clinical suspicion of FHM2. The avoidance of triggers and thus the prevention of attacks may be the most effective therapy for FHM2.     

Familial hemiplegic migraine with a PRRT2 mutation: Phenotypic variations and carbamazepine efficacy

ObjectiveTo understand the clinical characteristics of familial hemiplegic migraine (FHM) caused by a PRRT2 mutation and to examine the efficacy of preventive treatment.MethodsUsing the literature, we investigated clinical details of FHM in 3 generations of patients with a PRRT2 mutation and compared them with those in 17 patients with the same mutation from 6 families.ResultsIn most of the affected patients, the onset was observed during the teen years. Complicated phenotypes tended to be shared in each family, and five patients showed spontaneous remission. With regard to treatment, low-dose carbamazepine (CBZ) was effective in three patients.ConclusionConsidering the clinical features, we suggest that low-dose CBZ is efficacious for FHM treatment in patients with a PRRT2 mutation. The treatment duration should be carefully considered because some patients show spontaneous remission. More accumulated data from familial cases might help elucidate PRRT2 function and establish standard treatment for FHM.     

ATP1A2 gene mutations are not present in two sisters with basilar-type migraine associated with menses

Basilar-type migraine (BM) and hemiplegic migraine are clinically distinct subtypes of migraine with aura, however they do share clinical features and it is possible they may share genetic bases. In recent years, ATP1A2 and other gene mutations have been discovered in familial and sporadic hemiplegic migraine. More recently, an ATP1A2 mutation has been identified in an Italian family with BM. In this study we document the absence of ATP1A2 mutations in two Italian sisters with menstrual BM, suggesting that other genes are involved in the condition.     

A novel missense ATP1A2 mutation in a Finnish family with familial hemiplegic migraine type 2

Familial hemiplegic migraine (FHM), a rare autosomal dominant subtype of migraine with aura, has been linked to two chromosomal loci, 19p13 and 1q23. Mutations in the Na⁺,K⁺-ATPase 2 subunit gene, ATP1A2, on 1q23 have recently been shown to cause familial hemiplegic migraine type 2 (FHM2). We sequenced the coding regions of this gene in a Finnish chromosome 1q23-linked FHM family with associated symptoms such as coma and identified a novel A1033G mutation in exon 9. This mutation results in a threonine-to-alanine substitution at codon 345. This residue is located in a highly conserved N-terminal region of the M4–5 loop of the Na⁺,K⁺-ATPase. Furthermore, the T345A mutation co-segregated with the disorder in our family and was not present in 132 healthy Finnish control individuals. For these reasons it is most likely the FHM-causing mutation in this family.     

Is the CACNA1A gene involved in familial migraine with aura?

The discovery of mutations in the neural calcium channel (CACNA1A) gene in familial hemiplegic migraine (FHM), variant of migraine with aura, led to the suggestion that this gene might be involved in familial migraine with aura (FMA). We investigated whether the mutations in FHM are present in FMA patients, analyzing genomic DNA by PCR, single stranded conformation polymorphism, sequencing and restriction enzyme. No mutations were found. A known polymorphism (5682–14C>T) was found in exon 36. These findings suggest that the mutations found in FHM and the other known mutations of the CACNA1A gene are not the genetic basis of FMA. Genetic alterations in FMA patients may be localized on chromosome 19 but not in the CACNA1A exons we investigated. Received: 25 January 2002 / Accepted in revised form: 25 February 2002     

Loss and recovery of activities of alpha+ and alpha isozymes of (Na(+) + K+)-ATPase in cortical focal ischemia: GM1 ganglioside protects plasma membrane structure and function.

Alterations in cellular membrane structure and the subsequent failure of its function after CNS ischemia were monitored by analyzing changes in the plasma membrane marker enzyme (Na(+) + K(+)-ATPase. The levels of two isozymes of (Na(+) + K(+)-ATPase, alpha+ and alpha, which have distinct cellular and anatomical distributions, were studied to determine if differential cellular damage occurs in primary and peri-ischemic injury areas. The efficacy of monosialoganglioside (GM1) treatment was assessed, since this glycosphingolipid has been shown to reduce ischemic injury by protecting cell membrane structure/function. Using a rat model of cortical focal ischemia, levels of both ATPase isozyme activities were assayed in total membrane fractions from primary ischemic tissue (parietal cortex) and three peri-ischemic tissue areas (frontal, occipital, and temporal cortex) at 1, 3, 5, 7, and 14 days after ischemia. No significant loss of either isozyme's activity occurred in any tissue area at 1 day after ischemia. At 5 days, in the primary ischemic area, both isozyme activity levels decreased by 70-75%. The alpha+ enzyme activity loss persisted up to 14 days, while a 17% recovery in alpha activity occurred. In the three peri-ischemic tissue areas, enzyme activity losses ranged from 42%-59% at 3 days after ischemia. A complete restoration of both isozyme activities was seen at 14 days. After three days of GM1 ganglioside treatment there was no loss of total (Na*+) + K(+)-ATPase activity in the three peri-ischemic areas, and a significantly reduced loss in the primary infarct tissue. An autoradiographic analysis of brain coronal sections using 3H-ouabain supports the enzymatic data and GM1 effects. Reductions in 3H-ouabain binding in all cortical layers at 3 days after ischemia were visualized. GM1 treatment significantly reduced these 3H-ouabain binding losses. In summary, time-dependent quantitative changes in activity levels of ATPase isozymes (alpha+ and alpha) reflect the different degree of membrane damage that occurs in primary vs. peri-ischemic tissues (e.g., irreversible vs. reversible membrane damage), and that ischemia affects cell membranes of all neural elements in a largely similar fashion. GM1 ganglioside was found to reduce plasma membrane damage in all CNS cell types.     

Muscle phenotype in patients with myotonic dystrophy type 1

Introduction: The pathogenesis of muscle involvement in patients with myotonic dystrophy type 1 (DM1) is not well understood. In this study, we characterized the muscle phenotype in patients with confirmed DM1. Methods: In 38 patients, muscle strength was tested by hand‐held dynamometry. Myotonia was evaluated by a handgrip test and by analyzing the decrement of the compound muscle action potential. Muscle biopsies were assessed for morphological changes and Na⁺‐K⁺ pump content. Results: Muscle strength correlated with a decline in Na⁺‐K⁺ pump content (r = 0.60, P < 0.001) and with CTG expansion. CTG expansion did not correlate with severity of myotonia, proximal histopathological changes, or Na⁺‐K⁺ pump content. Histopathologically, we found few centrally placed nuclei (range 0.2–6.9%). Conclusions: The main findings of this study are that muscle weakness correlated inversely with CTG expansion and that central nuclei are not a prominent feature of proximal muscles in DM1. Muscle Nerve 47:409‐415, 2013     

Effect of neonatal hypothyroidism on the kinetic properties of Na+, K+ -ATPase from rat brain microsomes

The effects of neonatal hypothyroidism on the kinetic properties of Na+, K+ -ATPase from rat brain microsomes were examined. Neonatal hypothyroidism resulted in decreased Na+, K+ -ATPase activity compared to control samples (7.4 +/- 1.48 and 29.8 +/- 2.30 micromol Pi/h/mg protein, respectively, P < 0.001). Substrate kinetics studies with ATP, Na+ and K+ revealed that there were generalised decreases in Vmax. For ATP, Na+ and K+, activities resolved into two kinetic components in the control group. In hypothyroid animals, the low-affinity component for ATP was absent. The opposite pattern (i.e. an absence of the high-affinity component) was noted for Na+. For K+, although both kinetic components were discernible in neonatal hypothyroid brain microsomes, the Km of the high-affinity component was significantly higher (P < 0.001) compared to control samples. In the control group, the enzyme displayed allosteric behaviour at high concentrations of Mg2+; in hypothyroid animals, the pattern was completely allosteric. The Na+, K+ -ATPase enzyme from the hypothyroid brain microsomes bound two molecules of ATP rather than one, unlike in the control animals. Our results thus indicate that neonatal hypothyroidism results in an impairment of microsomal Na+, K+ -ATPase activity in the rat brain, together with subtle alterations in the kinetic properties of the enzyme.     

Epilepsy of infancy with migrating focal seizures: Six patients treated with bromide

PurposeWe present six patients with epilepsy of infancy with migrating focal seizures (EIMFS) and provide a comprehensive evaluation of potassium bromide therapy.MethodBetween February 1, 2007 and July 31, 2012, six patients who met the diagnostic criteria of EIMFS were treated with potassium bromide. Potassium bromide was added to other antiepileptic drugs (AEDs) in doses ranging from 30 to 80 mg/kg/day. Plasma bromide concentration was monitored. A therapeutic bromide concentration between 75 and 125 mg/dL was considered to be ideal.ResultsFour of six children responded well to bromide. One of these patients became seizure free, but remained severely mentally impaired. Two boys, currently 4 and 6 years of age, respectively, have monthly seizures as well as axial hypotonia and severe language impairment. The fourth child responded well to bromide, having only weekly seizures and moderate psychomotor retardation. The patient who became seizure free improved visual contact and head control. In the other three patients with good control, the seizures became focal without secondary generalization and status epilepticus and hospital admission was not required. The remaining two patients did not respond well to bromide. Adverse effects were seen in three cases: vomiting in one, drowsiness in another, and acneiform eruption in the face in the remaining patient. Adverse effects resolved with dose reduction.ConclusionEarly treatment with bromides should be considered in EIMFS to control the seizures and status epilepticus and to avoid progressive cognitive impairment. Potassium bromide is an old AED. Plasma concentration monitoring should be considered.     

Interactions of methylmercury with rat primary astrocyte cultures: inhibition of rubidium and glutamate uptake and induction of swelling

The ability of astrocytes to sequester MeHG may indicate an astrocyte-mediated role in MeHg's neurotoxicity. Hence, studies were undertaken to assess the effects of MeHg on metabolic functions in cultured astrocytes. MeHg (10⁻⁵ M) significantly inhibited the initial rate (5 min) of uptake of⁸⁶RbCl, used as a tracer for K⁺.⁸⁶RbCl uptake was also sensitive to the omission of medium Na⁺. MeHg (10⁻⁵ M) also markedly inhibited the initial rate of uptake (1 min) of the Na⁺-dependent uptake of [³H]l-glutamate. A second neurotoxin, MnCl₂ (0–5 × 10⁻⁴ M), did not alter [³H]glutamate or⁸⁶RbCl uptake. MeHg, but not MnCl₂, also stimulated the release of intracellular⁸⁶Rb⁺ in a dose-dependent fashion. This effect could be prevented by the administration of MeHg as the glutathione conjugate. These observations support the hypothesis that the astrocyte plasma membrane is an important target for MeHg's toxic effect and specifically that small concentrations of this organometal inhibit the ability of astrocytes to maintain a transmembrane K⁺ gradient. This would be expected to compromise the ability of astrocytes to control extracellular K⁺ either by spatial buffering or active uptake, resulting in cellular swelling. We therefore studied volume changes in astrocytes using uptake of [¹⁴C]3-O-methyl-d-glucose, in attached cells in response to exposure to MeHg. Exposure to MeHg (0–5 × 10⁻⁴ M) caused a marked increase in the cell volume that was proportional to concentrations of MeHg.     

Effect of Milacemide on Audiogenic Seizures and Cortical (Na+, K+)-ATPase of DBA/2J Mice

Milacemide (MLM, CP 1552 S, 2-N-pentylaminoacetamide), a glycinamide derivative, is currently being evaluated clinically for antiepileptic activity. Anticonvulsant properties have been shown in various animal models, but the mechanism of action of MLM is unclear. We studied its activity in audiogenic seizures of DBA/2J mice. MLM was effective in inhibiting the convulsions induced by sound with a biphasic dose-effect relation. The ED50 was 109 mg/kg orally against tonic extension. Higher doses were necessary to abolish clonic convulsion and running response. Because impaired cerebral (Na+, K+)-ATPase activity is supposed to play a role in epileptogenesis, we tested MLM on in vitro cortical enzymatic activity of DBA/2J mice. Basal (Na+, K+)-ATPase activity was unchanged by several concentrations of MLM in normal C57BL/6J and audiogenic DBA/2J mice. K+ activation (from 3 to 18 mM) of (Na+, K+)-ATPase is abolished in DBA/2J mice as compared with C57BL/6J mice, suggesting impaired glial (Na+, K+)-ATPase. In the presence of MLM (from 30 to 1000 mg/L), cortical (Na+, K+)-ATPase of DBA/2J mice is activated by high concentrations of K+, as in C57BL/6J mice. Results suggest that the antiepileptic activity of MLM in audiogenic mice may be secondary to an activation of a deficient glial (Na+, K+)-ATPase.     

Effects of naloxone and morphine on cerebral ischemia in gerbils

A therapeutic role for naloxone during stroke has been suggested, but a neurochemical site of action remains to be determined. Previous work with the gerbil cerebral cortex has shown that either bilateral secondary ischemia (60-min occlusion of the carotid arteries followed by 40-min reflow) or unilateral primary ischemia (permanent ligation of one carotid artery for 6 hr in symptomatic animals) produced deficits in both Na+, K+-ATPase (EC 3.6.1.3) activity and various parameters of activation of adenylate cyclase (EC 4.6.1.1). Pretreatment of gerbils with either naloxone or morphine failed to ameliorate or exacerbate, respectively, the neurological signs of ischemia; however, morphine did reduce mortality. Infusion of naloxone prior to ischemia afforded varying degrees of protection to forskolin, GTP analogs, and NE (norepinephrine) activation of adenylate cyclase, as well as to Na+, K+-ATPase (bilateral ischemia only). Similarly, morphine inhibited damage to basal activity of adenylate cyclase and to stimulation by NE, forskolin, and Gpp (NH)p (5'-guanylyl imidodiphosphate). Under in vitro conditions morphine increased the basal activity of adenylate cyclase but reduced responses to NE and forskolin. Furthermore, morphine injected into control gerbils elevated basal- and forskolin-elicited activities but reduced the activation of adenylate cyclase by NE.     

Neonatal status convulsivus, spongiform encephalopathy, and low activity of Na+/K(+)-ATPase in the brain.

The first and second child of a family died from neonatal seizures with no detectable brain malformation, metabolic, infectious, or chromosomal etiology. Neuropathological examination of the brain of the second child who died at 11 days revealed a widespread spongy state and a selective vulnerability of the astrocytes characterized by numerous enlarged bare astrocytic nuclei and different forms of astrocyte degeneration. The glial cells were strongly positive for glial fibrillary acidic protein and vimentin immunocytochemical reaction. Cortical measurement of Na+/K(+)-ATPase revealed very low enzyme activity. We hypothesize that a defect of Na+/K(+)-ATPase of the astrocytes could be the common pathogenetic factor for the congenital status convulsivus and for the spongy state.     

Effect of Na+, K(+)-ATPase modifiers on high-affinity ouabain binding determined by quantitative autoradiography.

There is growing evidence on the existence of endogenous ouabain-like factors that modulate Na+, K(+)-ATPase activity. In this laboratory, two soluble subfractions (peaks I and II) were previously separated from rat cerebral cortex, which had opposite effects on Na+, K(+)-ATPase activity. Peak I stimulated and peak II inhibited the enzyme (Rodríguez de Lores Arnaiz and Antonelli de Gómez de Lima, Neurochem Res 11:933-947, 1986). The same effects are now reported for K(+)-p-nitrophenyphosphatase activity. Localization of high-affinity ouabain binding in rat brain was done by quantitative autoradiography using a microcomputer digital imaging system. Peak I did not modify, whereas peak II blocked ouabain binding in areas 3-4 of cerebral cortex, dentate gyrus, stria terminalis, thalamic nuclei, and basal ganglia. Similar results were obtained when ouabain binding was determined in rabbit cerebral cortex and by a conventional filtration assay in nerve ending membranes obtained from rat cerebral cortex. These results favour the idea that the factor present in peak II fraction might behave as an ouabain-like substance.     

Insights into the pathogenesis of ATP1A1‐related CMT disease using patient‐specific iPSCs

The development of patient‐specific induced pluripotent stem cells (iPSCs) offered interesting insights in modeling the pathogenesis of Charcot‐Marie‐Tooth (CMT) disease and thus we decided to explore the phenotypes of iPSCs derived from a single CMT patient carrying a mutant ATP1A1 allele (p.Pro600Ala). iPSCs clones generated from CMT and control fibroblasts, were induced to differentiate into neural precursors and then into post‐mitotic neurons. Control iPSCs differentiated into neuronal precursors and then into post‐mitotic neurons within 6‐8 days. On the contrary, the differentiation of CMT iPSCs was clearly defective. Electrophysiological properties confirmed that post‐mitotic neurons were less mature compared to the normal counterpart. The impairment of in vitro differentiation of CMT iPSCs only concerned with the neuronal pathway, because they were able to differentiate into mesendodermal cells and other ectodermal derivatives. ATP1A1 was undetectable in the few neuronal cells derived from CMT iPSCs. ATP1A1 gene mutation (p.Pro600Ala), responsible for a form of axonal CMT disease, is associated in vitro with a dramatic alteration of the differentiation of patient‐derived iPSCs into post‐mitotic neurons. Thus, the defect in neuronal cell development might lead in vivo to a decreased number of mature neurons in ATP1A1‐CMT disease.     

Evidence that compromised K+ spatial buffering contributes to the epileptogenic effect of mutations in the human Kir4.1 gene (KCNJ10)

Mutations in the human Kir4.1 potassium channel gene (KCNJ10) are associated with epilepsy. Using a mouse model with glia-specific deletion of Kcnj10, we have explored the mechanistic underpinning of the epilepsy phenotype. The gene deletion was shown to delay K(+) clearance after synaptic activation in stratum radiatum of hippocampal slices. The activity-dependent changes in extracellular space volume did not differ between Kcnj10 mutant and wild-type mice, indicating that the Kcnj10 gene product Kir4.1 mediates osmotically neutral K(+) clearance. Combined, our K(+) and extracellular volume recordings indicate that compromised K(+) spatial buffering in brain underlies the epilepsy phenotype associated with human KCNJ10 mutations.