WNK1/HSN2 founder mutation in patients with hereditary sensory and autonomic neuropathy: A Japanese cohort study.

The clinical and genetic spectrum of hereditary sensory and autonomic neuropathy (HSAN) is still unknown in Japan. We collected a broad cohort of 33 unrelated patients with predominant sensory and/or autonomic dysfunctions, who were referred to our genetic laboratory. A gene panel sequencing targeting 18 HSAN-related genes was performed using a next-generation sequencing system. A recurrent frame shift mutation in the WNK1/HSN2 gene, c.3237_3238insT (p.Asp1080*), was detected in 5 patients. This mutation was homozygous in 4 cases and of a compound heterozygous genotype in 1 case. Geographic and haplotype analysis of all 5 patients suggested a founder event. In addition, a novel heterozygous nonsense variant, c.2615C>G (p.Ser872*), was identified. All the 5 patients presented with severe sensory and autonomic dysfunctions at birth or during adolescence. In 2 patients, an uncommon phenotype of acute pathological pain presented at ~50 years of age. Here, we present the first founder mutation of WNK1/HSN2, in addition to French Canadian, which accounts for ~15.2% of Japanese patients with HSAN in our cohort. We have also reviewed all previously described mutations in WNK1/HSN2 and reconciled their nomenclature strategy on the basis of the current longest transcript.

Aberrantly spliced alpha-dystrobrevin alters alpha-syntrophin binding in myotonic dystrophy type 1.

BACKGROUND: Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG repeat expansion in the DMPK gene. Aberrant messenger RNA (mRNA) splicing of several genes has been reported to explain some of the symptoms in DM1, but the cause of muscle wasting is still unknown. By contrast, many forms of muscular dystrophy are caused by abnormalities of the dystrophin-glycoprotein complex (DGC). alpha-Dystrobrevin is a key component of the DGC in striated muscle and plays important roles in maturation and signal transduction by interacting with alpha-syntrophin. The goal of this study was to investigate alternative splicing of alpha-dystrobrevin in DM1 and examine alpha-syntrophin binding of different alpha-dystrobrevin splice isoforms. METHODS: Splicing patterns of alpha-dystrobrevin in DM1 muscle were studied by reverse-transcriptase PCR. Expression of the variant splice isoform was examined by immunoblotting and immunohistochemistry. Alternatively spliced isoforms were expressed in cultured cells to investigate interaction with alpha-syntrophin. alpha-Syntrophin expression was examined by immunoblotting. RESULTS: alpha-Dystrobrevin mRNA including exons 11A and 12 was increased in both skeletal and cardiac muscle of DM1 patients. The aberrantly spliced alpha-dystrobrevin isoform was localized to the sarcolemma, and showed increased binding with alpha-syntrophin. Furthermore, levels of alpha-syntrophin associated with the DGC were increased in DM1 muscle. CONCLUSION: Alternative splicing of alpha-dystrobrevin is dysregulated in myotonic dystrophy type 1 (DM1) muscle, resulting in changes in alpha-syntrophin binding. These results raise the possibility that effects on alpha-dystrobrevin splicing may influence signaling in DM1 muscle cells.

Beneficial effect of interferon-beta treatment in patients with multiple sclerosis is associated with transient increase in serum IL-6 level in response to interferon-beta injection.

In order to predict the clinical benefit of interferon-beta (IFN-beta) to patients with multiple sclerosis (MS), the following markers were investigated; (1) chronological change of cytokines (IFN-gamma, TNF-alpha, IL-6, IL-10, and TGF-beta) after administration of IFN-beta, (2) untoward effects of IFN-beta such as headache and arthralgia, (3) backgrounds of the patients such as age and relapse rate, (4) efficacy of IFN-beta therapy assessed by the change of relapse rate and progression of disability. Chronological blood sampling was performed 0, 10, and 24 h after injection of IFN-beta. The increase of serum IL-6 level in response to IFN-beta administration was associated with headache, arthralgia, relapse rate before treatment, and disability score at the initiation of the therapy. Significant association of change of serum TNF-alpha with age and headache was also observed. The important finding in this study was that patients with a transient increase in IL-6 in response to IFN-beta showed a slow disease progression. This result suggests that this transient increase in the serum IL-6 predicts favorable response to IFN-beta treatment.

Mexiletine block of disease-associated mutations in S6 segments of the human skeletal muscle Na(+) channel.

1. Over twenty different missense mutations in the alpha-subunit of the adult skeletal muscle Na(+) channel (hSkM1) have been identified as a cause of myotonia or periodic paralysis. We examined state-dependent mexiletine block for mutations involving the putative binding site in S6 segments (V445M, S804F, V1293I, V1589M and M1592V). Whole-cell Na(+) currents were measured from wild-type (WT) and mutant channels transiently expressed in HEK cells. 2. Use-dependent block (10 ms pulses to -10 mV, at 20 Hz) in 100 microM mexiletine was reduced modestly by mutations in IVS6 (V1589M, M1592V) and enhanced by the mutation in IS6 (V445M). For mutations in IIS6 (S804F) and IIIS6 (V1293I) use-dependent block was not statistically different from that of wild-type channels. 3. Resting-state block (10 ms pulses to -10 mV from -150 mV, at 0.1 Hz) of S6 mutants was comparable to that of WT (dissociation constant for resting channels, K(R) = 650 +/- 40 microM, n = 9). The S6 mutant with the greatest change in K(R) was V445M (K(R) = 794 +/- 45 microM, n = 5), but this difference was only marginally significant (P = 0.047). 4. A modified technique for estimating local anaesthetic affinity of inactivated channels was developed to reduce errors due to slow inactivation and to failure of drug binding to reach equilibrium. Mexiletine affinity for inactivated channels was reduced by mutations in IVS6 (V1589M: dissociation constant for the inactivated state (K(I)) = 44.7 microM; M1592V: K(I) = 40.0 microM) and increased by the mutation in IS6 (V445M: K(I) = 15.0 microM), compared to wild-type channels (K(I) = 28.3 microM). 5. We conclude that the disease-associated S6 mutations in domains I-IV cause at most a 2-fold change in inactivated state affinity and have even less of an effect on resting block. Model simulations show that the reduced use-dependent block of IVS6 mutants derives primarily from an increased off-rate at hyperpolarized potentials, whereas the enhanced use-dependent block of the IS6 mutant was due to a higher affinity for inactivated V445M channels.

A new mutation in a family with cold-aggravated myotonia disrupts Na(+) channel inactivation.

OBJECTIVE: To identify the molecular and physiologic abnormality in familial myotonia with cold sensitivity, hypertrophy, and no weakness. BACKGROUND: Sodium channel mutations were previously identified as the cause of several allelic disorders with varying combinations of myotonia and periodic paralysis. A three-generation family with dominant myotonia aggravated by cooling, but no weakness, was screened for mutations in the skeletal muscle sodium channel alpha-subunit gene (SCN4A). METHODS: Single-strand conformation polymorphism was used to screen all 24 exons of SCN4A and abnormal conformers were sequenced to confirm the presence of mutations. The functional consequence of a SCN4A mutation was explored by recording sodium currents from human embryonic kidney cells transiently transfected with an expression construct that was mutated to reproduce the genetic defect. RESULTS: A three-generation Italian family with myotonia is presented, in which a novel SCN4A mutation (leucine 266 substituted by valine, L266V) is identified. This change removes only a single methylene group from the 1,836-amino-acid protein, and is present in a region of the protein previously not known to be critical for channel function (domain I transmembrane segment 5). Electrophysiologic studies of the L266V mutation showed defects in fast inactivation, consistent with other disease-causing SCN4A mutations studied to date. Slow inactivation was not impaired. CONCLUSIONS: This novel mutation of the sodium channel indicates that a single carbon change in a transmembrane alpha-helix of domain I can alter channel inactivation and cause cold-sensitive myotonia.

The expression of ion channel mRNAs in skeletal muscles from patients with myotonic muscular dystrophy.

We investigated gene expression patterns of ion channels including the apamin-sensitive small-conductance Ca(2+)-activated K(+) (SK3) channel, the adult isoform of the skeletal muscle Na(+) channel (SkM1), the fetal isoform of skeletal muscle Na(+) channel (H1), and the Cl(-) channel (ClC-1) by using the semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for muscle samples from patients with adult onset myotonic dystrophy (DM), amyotrophic lateral sclerosis, and polymyositis. Patients with DM showed a significant increase in SK3 mRNA but not in mRNAs for other ion channels. The increased expression of SK3 gene in DM did not correlate with H1, the marker of muscle denervation, or the percentage of type 2C fiber, the marker of muscle regeneration.

Calcium increase in mouse skeletal muscles by triparanol: a drug to induce myotonic dystrophy-like clinical manifestations.

Triparanol (Trp) is known to cause clinical features similar to those seen in myotonic dystrophy, including myotonia, cataract and baldness. To explore the pathophysiological mechanism of myotonic dystrophy, we examined the effect of Trp on intracellular calcium in cultured skeletal myoblasts and myotubes as well as cardiac myocytes by using a fluorescent indicator. Trp preferentially induced increase of intracellular calcium in myotubes of skeletal muscles. Since the increase of calcium was inhibited by thapsigargin pretreatment but not by extracellular calcium elimination, it appears that triparanol might act mostly on intracellular calcium stores. Trp also inhibited the increase of calcium in myotubes induced by acetylcholine. Trp might cause myotonia possibly through the increase of intracellular calcium from intracellular stores.

Enhanced slow inactivation by V445M: a sodium channel mutation associated with myotonia.

Over 20 different missense mutations in the alpha subunit of the adult skeletal muscle Na channel have been identified in families with either myotonia (muscle stiffness) or periodic paralysis, or both. The V445M mutation was recently found in a family with myotonia but no weakness. This mutation in transmembrane segment IS6 is novel because no other disease-associated mutations are in domain I. Na currents were recorded from V445M and wild-type channels transiently expressed in human embryonic kidney cells. In common with other myotonic mutants studied to date, fast gating behavior was altered by V445M in a manner predicted to increase excitability: an impairment of fast inactivation increased the persistent Na current at 10 ms and activation had a hyperpolarized shift (4 mV). In contrast, slow inactivation was enhanced by V445M due to both a slower recovery (10 mV left shift in beta(V)) and an accelerated entry rate (1.6-fold). Our results provide additional evidence that IS6 is crucial for slow inactivation and show that enhanced slow inactivation cannot prevent myotonia, whereas previous studies have shown that disrupted slow inactivation predisposes to episodic paralysis.

Lambert-Eaton syndrome antibodies inhibit acetylcholine release and P/Q-type Ca2+ channels in electric ray nerve endings.

1. The types of voltage-dependent calcium channels (VDCCs) present in the cholinergic terminals isolated from the electric organ of the ray, Narke japonica, were characterized on the basis of their pharmacological sensitivity to specific antagonists. Inhibition of these channel types by autoantibodies from patients with the Lambert-Eaton syndrome (LES) was then studied to determine the specificity of the pathogenic IgG. 2. In normal untreated synaptosomal preparations, maximal doses of N- and P and/or Q-type Ca2+ channel antagonists, omega-conotoxin GVIA and omega-agatoxin IVA, inhibited depolarization-evoked ACh release by 47 % and 43 %, respectively. Calciseptine, an L-type VDCC antagonist, caused a 20 % reduction in the release. This indicates that the exocytotic release process is predominantly mediated by N- and P/Q-type VDCCs. 3. LES IgG or sera caused an inhibition of ACh release by 39-45 % in comparison with the control antibody-treated preparations. The ionomycin-induced ACh release, however, was not altered by the antibodies. Additionally, the same LES antibodies inhibited whole-cell calcium currents (ICa) in bovine adrenal chromaffin cells. Thus, the pathogenic antibodies exert their action on VDCCs present in the synaptosomes. 4. The efficacy of three Ca2+ channel antagonists in blocking ACh release was determined in preparations pretreated with LES IgG. omega-Agatoxin IVA produced only an additional 3-5 % reduction in release beyond that obtained with LES antibodies. Despite the pretreatment with LES IgG, omega-conotoxin GVIA and calciseptine inhibited the release to nearly their control levels. 5. These results indicate that LES antibodies mainly downregulate P/Q-type Ca2+ channels which contribute to presynaptic transmitter release from the cholinergic nerve terminals of electric organ. 6. The present findings are consistent with the hypothesis that P/Q-type VDCCs at the neuromuscular junction are the target of LES antibodies and that their inhibition by the antibodies produces the characteristic neuromuscular defect in this disease.

[A case of Kallmann syndrome with empty sella and arachnoid cyst].

We report a 28-year-old man of Kallmann syndrome with arachnoid cyst and empty sella. At age 22, he was admitted with acute slipped capital epiphysis and diagnosed as primary hypogonadotropinemia, because of no response to LH-RH before and after 7-day LH-RH injection. He was treated with androgen for only one year. On his second admission due to femoral head necrosis at age 28, the endocrinological evaluation suggested hypothalamic hypogonadotropinemia. Although he had mild hyposomia, we diagnosed him as Kallmann syndrome, because abnormalities of rhinencephalon was present on MRI. Arachnoid cyst in the middle cranial fossa and empty sella were also observed on MRI and the ballooning of the sella had been advanced on plain X-ray for these 6 years. As Kallmann syndrome is known to be accompanied with midline craniofacial anomalies, the dysplasia of sellar diaphragm might be originated by the same pathogenesis. In this case, empty sella might be caused by impaired CSF dynamics due to arachnoid cyst as well as possible constitutional anomaly of the diaphragm.

[The effects of spinal fusion on respiratory function and quality of life in Duchenne muscular dystrophy].

We studied the functional outcome of spinal fusion for the surgical treatment of scoliosis in 8 patients with Duchenne muscular dystrophy (DMD). The mean age of DMD patients at the time of the surgery and the mean follow-up duration was 13.8 (12.3 to 15.4) and 3.9 (1.5 to 6.8) years, respectively. The average spinal angle (Cobb angle) was corrected from 58.8 to 28.6 degrees with the mean corrective rate of 51.3% by the surgical intervention. The correction rate was higher and the corrected Cobb angle remained unchanged during follow-up period in mildly scoliotic patients. Forced vital capacity (FVC) increased post-operatively in 3 patients with moderate scoliosis (Cobb angle: 50 to 80 degrees), indicating that the correction of spinal alignment is effective for the treatment of decreased thoracic volume in DMD. On the other hand, two cases with low % FVC (16.9% and 30.4%, respectively) had poor prognosis in respiratory status. Namely, one died of pneumonia at 17 months after the surgery and the other required mechanical ventilation via nasal mask at 3 years post surgery. Sitting balance improved in all patients, which resulted in more functional use of their upper extremities. During the follow-up period, all patients except one patient who died of pneumonia could maintain sitting balance without support. Moreover these included 2 patients over 20 year old. No complications related to spinal deformities have been found in these patients. Previous study in our hospital showed that 7 of 48 (14.6%) of DMD patients spent all their lives without apparent scoliosis (Cobb angle less than 30 degrees). These suggest that spinal fusion could be recommended for patients with Cobb angle more than 30 degrees and with % FVC more than 35%. Although the impact of spinal fusion upon the life expectancy remains unclear, favorable effect on respiratory function and quality of life can be expected for carefully selected patients with DMD.

[A case of idiopathic spinal cord herniation].

The authors experienced a case of idiopathic spinal cord herniation with duplicated dura mater. A 63-year-old woman presented right dominant slowly progressive spastic paraplegia and dissociated sensory disturbance. Magnetic resonance imaging (MRI) demonstrated an enlarged dorsal arachnoid space associated with an apparently focally narrowed thoracic cord. The cord was kinked towards the anterior and closely applied to vertebral body at the level of Th3-4. Computed tomographic myelography (CTM) revealed homogeneous filling at dorsal arachnoid space immediately after injection and no defects. At operation multilocular arachnoid cyst and duplicated dura mater was respectively observed dorsally, and ventrally. From defected area of the inner layer, a ventral part of the spinal cord was incarcerated between the two dural layers. After rejection of arachnoid cyst and inner layer was performed, the patient recovered neurologically. Idiopathic spinal cord herniation is a rare disease that shows slowly progressive myelopathy at middle age. The herniations were observed at ventral thoracic cord in all reported cases. The mechanism of this disease is still uncertain. But at least three successive factors seem to be necessary for formation of herniation, 1) abnormal structure of the dura mater such as defect, diverticulum and duplication; 2) adhesion between the cord and the destructive dura mater, and 3) continuous cerebrospinal fluid (CSF) pressure pushing the cord outward from subdural space. In the thoracic spine, mobility is limited compared with the cervical and lumbar spine, and because of physiological curvature the cord situates rather ventrally. For these reasons the incidence of adhesion might be higher at ventral thoracic spine. Although neuroradiological imagings especially MRI and CTM were useful, operative findings were necessary for definitive diagnosis in many reported cases. Considering the effectiveness of surgical treatment, study of the ventral side of the cord should be important to avoid misdiagnosis.

Synaptic input-induced increase in intraneuronal Ca2+ in the medial vestibular nucleus of young rats.

In the medial vestibular nucleus (MVN), an input-dependent influx of Ca2+ into neurons through N-methyl-D-aspartate (NMDA) receptor-linked channels and/or voltage-dependent Ca2+ channels is suggested as underlying certain mechanisms of plasticity of the vestibular system. To see whether there is an increase in intracellular Ca2+ induced by afferent synaptic inputs to MVN neurons, we measured changes in [Ca2+]i with microfluorometry using a Ca2+ indicator, rhod-2, following electrical stimulation of ipsilateral vestibular afferents and commissural fibers in slice preparations of the brainstem of young rats (4-7 days postnatal). Single shock stimulation of ipsilateral afferents or commissural fibers induced an increase in fluorescence intensity lasting for several seconds. An application of 2-amino-5-phosphonovaleric acid (APV), an antagonist of NMDA receptors, almost completely blocked this stimulus-induced rise in fluorescence intensity. Nifedipine, an L-type Ca2+ channel blocker, also reduced the stimulus-induced rise in fluorescence intensity to 44-51% of the control value. These results suggest that synaptic inputs from the afferent and commissural pathways induce an influx of Ca2+ into MVN neurons due, at least in part, to the activation of NMDA receptors and the subsequent operation of L-type Ca2+ channels in young rats.

Induction of LTD but not LTP through metabotropic glutamate receptors in visual cortex.

Long-term potentiation (LTP) and long-term depression (LTD), often used as essential components in synaptic models for learning, memory and forgetting, can be produced in cortical tissue by repetitive activation of neural pathways under different stimulus conditions. The involvement of metabotropic glutamate receptors (mGluRs) has been postulated to be necessary for the establishment of either or both forms of synaptic plasticity in hippocampus. The recent introduction of a specific antagonist for mGluRs, (+/-)-alpha-methyl-4-carboxyphenylglycine, prompted the investigation of the respective involvement of this receptor population in the induction of LTP and LTD in visual cortex of the rat in vitro. The results suggest the critical involvement of mGluRs in producing LTD but not LTP.

Intracellular calcium increase induced by GABA in visual cortex of fetal and neonatal rats and its disappearance with development.

To address the question of whether gamma-aminobutyric acid (GABA) induces a change in the concentration of Ca2+ in neurons of the developing visual cortex, and if so, to elucidate a developmental profile of such a GABA-induced change, we measured intracellular Ca2+ signals using microscopic fluorometry in visual cortical slices loaded with rhod-2. The slices were prepared from rat fetuses of embryonic day 18 (E18) and rat pups of postnatal days 0-30 (P0-P30). Application of GABA through the perfusate at 100 microM induced a marked rise in intracellular Ca2+ signals in the cortical plate and subplate at E18 and P0-P2. After P5 the GABA-induced rise in Ca2+ dramatically reduced, and at P20 and thereafter it became undetectable. At E18 and P0-P2 an agonist for GABAA receptor, muscimol, induced a Ca2+ rise in the same way as did GABA, while a GABAB receptor agonist, baclofen, did not induce any significant rise in Ca2+ signals. Also, a GABAA receptor antagonist, bicuculline, blocked the GABA-induced rise in Ca2+ signals. These results indicate that the Ca2+ rise is triggered by activation of GABAA receptors. The application of Ni2+ at a concentration high enough to block all types of voltage-dependent CA2+ channels prevented the Ca2+ signals from increasing in response to GABA application, suggesting that Ca2+ may be influxed through such channels following depolarization evoked by GABA.

Laminar difference in tetanus-induced increase of intracellular Ca2+ in visual cortex of young rats.

Changes in intracellular Ca2+ evoked by electrical stimulation of the white matter were observed by means of microfluorometry with a Ca2+ indicator, rhod-2, in slice preparations of the visual cortex obtained from young rats. Tetanic stimulation at 5 Hz for 1 min induced a marked fluorescence increase, while single-shock stimulation did not induce a sizable increase in normal perfusate. The tetanus-induced increase took place in a column-like manner from layer VI near the stimulation site to layer II/III of the cortex, although it spread horizontally in layer II/III. The magnitude of fluorescence rise was largest in layer II/III of the cortex. Since N-methyl-D-aspartate (NMDA) receptors are known to exist only on neurons, the following results are taken to indicate that the fluorescent signal is derived mostly from postsynaptic neurons: Application of NMDA in the presence of tetrodotoxin induced a marked fluorescence increase with the same laminar bias as tetanic stimulation did, and the fluorescence increase by single-shock stimulation in Mg(2+)-free medium was almost completely blocked by an antagonist for NMDA receptors. These results support the hypothesis that input-associated entry of Ca2+ into postsynaptic neurons triggers processes for induction of long-term potentiation of synaptic efficacy.

Contribution of NMDA receptors to tetanus-induced increase in postsynaptic Ca2+ in visual cortex of young rats.

Mechanisms underlying the Ca2+ increase during tetanic synaptic inputs in layer II/III of visual cortical slices of young rats were investigated with microfluorometry using a Ca2+ indicator, rhod-2, and simultaneous recordings of field potentials evoked by white matter stimulation. Application of an antagonist for N-methyl-D-aspartate (NMDA) receptors, 2-amino-5-phosphonovalerate, did not significantly affect field potentials but reduced the tetanus-induced fluorescence rise to 56%, on average, of the control values. Application of a broad-spectrum antagonist for both NMDA and non-NMDA receptors, kynurenate, completely abolished the synaptically evoked component of field potentials and decreased the tetanus-induced fluorescence rise to 42%. Application of a non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, in the Mg(2+)-free medium diminished the field potentials but did not decrease the tetanus-induced fluorescence increase. Nifedipine and diltiazem, L-type Ca2+ channel blockers, and Ni2+, a relatively selective blocker for T-type Ca2+ channels, did not affect the tetanus-induced fluorescence rise. These results indicate that NMDA receptors play a significant role in the increase of intracellular Ca2+ during tetanic synaptic inputs in the visual cortex of young rats.