Ibi T

Open-label trial and randomized, double-blind, placebo-controlled, crossover trial of hydrogen-enriched water for mitochondrial and inflammatory myopathies

BackgroundMolecular hydrogen has prominent effects on more than 30 animal models especially of oxidative stress-mediated diseases and inflammatory diseases. In addition, hydrogen effects on humans have been reported in diabetes mellitus type 2, hemodialysis, metabolic syndrome, radiotherapy for liver cancer, and brain stem infarction. Hydrogen effects are ascribed to specific radical-scavenging activities that eliminate hydroxyl radical and peroxynitrite, and also to signal-modulating activities, but the detailed molecular mechanisms still remain elusive. Hydrogen is a safe molecule that is largely produced by intestinal bacteria in rodents and humans, and no adverse effects have been documented.MethodsWe performed open-label trial of drinking 1.0 liter per day of hydrogen-enriched water for 12 weeks in five patients with progressive muscular dystrophy (PMD), four patients with polymyositis/dermatomyositis (PM/DM), and five patients with mitochondrial myopathies (MM), and measured 18 serum parameters as well as urinary 8-isoprostane every 4 weeks. We next conducted randomized, double-blind, placebo-controlled, crossover trial of 0.5 liter per day of hydrogen-enriched water or placebo water for 8 weeks in 10 patients with DM and 12 patients with MM, and measured 18 serum parameters every 4 weeks.ResultsIn the open-label trial, no objective improvement or worsening of clinical symptoms was observed. We, however, observed significant effects in lactate-to-pyruvate ratios in PMD and MM, fasting blood glucose in PMD, serum matrix metalloproteinase-3 (MMP3) in PM/DM, and serum triglycerides in PM/DM. In the double-blind trial, no objective clinical effects were observed, but a significant improvement was detected in lactate in MM. Lactate-to-pyruvate ratios in MM and MMP3 in DM also exhibited favorable responses but without statistical significance. No adverse effect was observed in either trial except for hypoglycemic episodes in an insulin-treated MELAS patient, which subsided by reducing the insulin dose.ConclusionsHydrogen-enriched water improves mitochondrial dysfunction in MM and inflammatory processes in PM/DM. Less prominent effects with the double-blind trial compared to the open-label trial were likely due to a lower amount of administered hydrogen and a shorter observation period, which implies a threshold effect or a dose-response effect of hydrogen.

Clinical features and a mutation with late onset of limb girdle muscular dystrophy 2B

Objective and methods Dysferlin encoded by DYSF deficiency leads to two main phenotypes, limb girdle muscular dystrophy (LGMD) 2B and Miyoshi myopathy. To reveal in detail the mutational and clinical features of LGMD2B in Japan, we observed 40 Japanese patients in 36 families with LGMD2B in whom dysferlin mutations were confirmed. Results and conclusions Three mutations (c.1566C>G, c.2997G>T and c.4497delT) were relatively more prevalent. The c.2997G>T mutation was associated with late onset, proximal dominant forms of dysferlinopathy, a high probability that muscle weakness started in an upper limb and lower serum creatine kinase (CK) levels. The clinical features of LGMD2B are as follows: (1) onset in the late teens or early adulthood, except patients homozygous for the c.2997G>T mutation; (2) lower limb weakness at onset; (3) distal change of lower limbs on muscle CT at an early stage; (4) impairment of lumbar erector spinal muscles on muscle CT at an early stage; (5) predominant involvement of proximal upper limbs; (6) preservation of function of the hands at late stage; (7) preservation of strength in neck muscles at late stage; (8) lack of facial weakness or dysphagia; (9) avoidance of scoliosis; (10) hyper-Ckaemia; (11) preservation of cardiac function; and (12) a tendency for respiratory function to decline with disease duration. It is important that the late onset phenotype is found with prevalent mutations.

Four parameters increase the sensitivity and specificity of the exon array analysis and disclose 25 novel aberrantly spliced exons in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is an RNA gain-of-function disorder in which abnormally expanded CTG repeats of DMPK sequestrate a splicing trans-factor MBNL1 and upregulate another splicing trans-factor CUGBP1. To identify a diverse array of aberrantly spliced genes, we performed the exon array analysis of DM1 muscles. We analyzed 72 exons by RT-PCR and found that 27 were aberrantly spliced, whereas 45 were not. Among these, 25 were novel and especially splicing aberrations of LDB3 exon 4 and TTN exon 45 were unique to DM1. Retrospective analysis revealed that four parameters efficiently detect aberrantly spliced exons: (i) the signal intensity is high; (ii) the ratio of probe sets with reliable signal intensities (that is, detection above background P-value=0.000) is high within a gene; (iii) the splice index (SI) is high; and (iv) SI is deviated from SIs of the other exons that can be estimated by calculating the deviation value (DV). Application of the four parameters gave rise to a sensitivity of 77.8% and a specificity of 95.6% in our data set. We propose that calculation of DV, which is unique to our analysis, is of particular importance in analyzing the exon array data.

Functional characterisation of mitochondrial tRNA(Tyr) mutation (5877-->GA) associated with familial chronic progressive external ophthalmoplegia.

Editor—Chronic progressive external ophthalmoplegia (CPEO) is a common clinical manifestation of mitochondrial cytopathies characterised by ophthalmoplegia and ptosis.1 Approximately two-thirds of CPEO patients harbour a large, heteroplasmic, mitochondrial DNA (mtDNA) deletion.2 Some other CPEO patients carry a point mutation in the mitochondrial tRNA genes. Twelve point mutations in six mitochondrial tRNA genes have been reported to date in association with CPEO (Mitomap at http://www.gen.emory.edu/mitomap.html). Among the 12 mutations, 5703C→T in the tRNAAsn gene has been functionally characterised.3 Here we report functional analysis of 5877G→A in tRNATyr identified in a patient with CPEO4 using ρ cells that lack mtDNA. A 45 year old woman had moderate degrees of ptosis, external ophthalmoplegia, and proximal muscle weakness from the age of 28. She had no sensorineural hearing loss, ataxia, pigmentary retinopathy, hypogonadism, or mental retardation. She had episodic diarrhoea of unknown aetiology. An ECG showed atrioventricular conduction block, while EEG, brain CT, and brain MRI showed no abnormalities. An exercise loading test of 15 watts for 15 minutes on a bicycle ergometer5 raised her serum lactate from 6.9 mg/dl to 24.0 mg/dl (normal, less than 18.0 mg/dl), and her serum pyruvate from 0.6 mg/dl to 1.4 mg/dl (normal, less than 1.3 mg/dl), thereby increasing the lactate to pyruvate ratio from 10.7 to 17.1 (normal, less than 13.8). A biopsy specimen obtained from the biceps brachii showed 4.0% ragged red fibres and 0.7% cytochrome …

Direct DNA sequencing from colony: analysis of multiple deletions of mitochondrial genome

We have developed a rapid and efficient nucleotide sequencing technique, named the colony direct sequencing method, which combines both the conventional cloning method for picking up a single gene and the polymerase chain reaction (PCR) method for amplifying the gene directly from a colony. In the present study, the colony direct PCR product was used both for identification of the DNA insert and for nucleotide sequencing by an automated DNA analysis system. A nucleotide sequence of 300 to 400 bp could be determined within 13 h after picking the bacterial colonies on LB medium plates. We applied this method to sequencing of junctional regions of multiple deleted mtDNAs in two siblings with inherited recurrent myoglobinuria. Mitochondrial DNA fragments with deletions were amplified by PCR and then cloned into plasmids. Among 48 white colonies propagated on LB medium plates, nine different clones were identified by PCR directly from colonies. Determination of six different junctional sequences disclosed involvement of directly repeated sequences of 2 to 12 bp in length on each side of the deletions. We believe that the colony direct sequencing method will be a powerful tool in molecular genetics for identification of a single gene among polymorphic DNAs.

Immunohistochemical demonstration of carbonic anhydrase III and muscle-specific enolase in paraffin-embedded human skeletal muscle sections

SummaryWe have demonstrated the histochemical fiber types of human skeletal muscle in paraffin sections by the immunohistochemical method, together with compatible observations previously made in frozen sections that carbonic anhydrase III is mainly localized in type 1 fibers (Shima et al. 1983), and muscle-specific enolase in type 2 fibers (Ibi et al. 1983). This method is useful to analyze the fiber types when frozen muscle samples at biopsy or autopsy cannot be obtained.

LDB3 splicing abnormalities are specific to skeletal muscles of patients with myotonic dystrophy type 1 and alter its PKC binding affinity.

Myotonic dystrophy type 1 (DM1) is caused by transcription of CUG repeat RNA, which causes sequestration of muscleblind-like 1 (MBNL1) and upregulation of CUG triplet repeat RNA-binding protein (CUG-BP1). In DM1, dysregulation of these proteins contributes to many aberrant splicing events, causing various symptoms of the disorder. Here, we demonstrate the occurrence of aberrant splicing of LIM domain binding 3 (LDB3) exon 11 in DM1 skeletal muscle. Exon array surveys, RT-PCR, and western blotting studies demonstrated that exon 11 inclusion was DM1 specific and could be reproduced by transfection of a minigene containing the CTG repeat expansion. Moreover, we found that the LDB3 exon 11-positive isoform had reduced affinity for PKC compared to the exon 11-negative isoform. Since PKC exhibits hyperactivation in DM1 and stabilizes CUG-BP1 by phosphorylation, aberrant splicing of LDB3 may contribute to CUG-BP1 upregulation through changes in its affinity for PKC.

A sodium channel myotonia due to a novel SCN4A mutation accompanied by acquired autoimmune myasthenia gravis

Mutations of the voltage gated sodium channel gene (SCN4A) are responsible for non-dystrophic myotonia including hyperkalemic periodic paralysis, paramyotonia congenita, and sodium channel myotonia, as well as congenital myasthenic syndrome. In vitro functional analyses have demonstrated the non-dystrophic mutants to show a gain-of-function defect of the channel; a disruption of fast inactivation, an enhancement of activation, or both, while the myasthenic mutation presents a loss-of function defect. This report presents a case of non-dystrophic myotonia that is incidentally accompanied with acquired myasthenia. The patient presented a marked warm-up phenomenon of myotonia but the repeated short exercise test suggested mutations of the sodium channel. The genetic analysis identified a novel mutation, G1292D, of SCN4A. A functional study of the mutant channel revealed marked enhancement of activation and slight impairment of fast inactivation, which should induce muscle hyperexcitability. The effects of the alteration of channel function to the myasthenic symptoms were explored by using stimulation of repetitive depolarization pulses. A use-dependent channel inactivation was reduced in the mutant in comparison to normal channel, thus suggesting an opposing effect to myasthenia.

MM1-type sporadic Creutzfeldt-Jakob disease with unusually prolonged disease duration presenting with panencephalopathic-type pathology.

We report an autopsy case of MM1‐type sporadic Creutzfeldt‐Jakob disease (sCJD) with an unusually prolonged disease duration of 58 months. The initial symptom was progressive mental disorder followed by advanced cognitive impairment. Clinical progression was generally slow; myoclonus appeared at 17 months and periodic sharp‐wave complexes on electroencephalogram at 21 months. A state of akinetic mutism occurred 29 months after the onset of symptoms. MRI showed gradually progressive cerebral atrophy. Neuropathologic examination showed widespread severe brain involvement. In the cerebral neocortex, widespread severe tissue rarefaction, hypertrophic astrocytosis and neuron loss (so‐called status spongiosus) were observed. The cerebral white matter showed diffuse myelin pallor with intense hypertrophic astrocytosis, numerous foamy macrophages and emperipolesis, indicating panencephalopathic‐type sCJD pathology. The brainstem was relatively preserved from sCJD pathology, with the exception of the pontine nucleus and pyramidal tract. This may explain the prolonged disease duration without respiratory insufficiency until the terminal stage. Immunohistochemistry for prion protein (PrP) showed widespread synaptic‐type PrP deposits in the cerebral neocortex, hippocampus and thalamus. The striatum and cerebellar cortex showed faint synaptic‐type PrP deposition with some areas of small plaque‐like PrP deposition. Sparse PrP deposition was also observed in the brainstem. Analysis of the PrP gene showed no mutation but methionine homozygosity at polymorphic codon 129. Western blot analysis of protease‐resistant PrP indicated type 1 PrP. To our knowledge, this is the longest reported disease duration of MM1‐type sCJD.

Dyskinesia-hyperpyrexia syndromeを合併したパーキンソン病の1例

Non-physiological, excessive dopaminergic stimulation can cause dyskinesia-hyperpyrexia syndrome (DHS), which was initially reported by Gil-Navarro and Grandas in 2010. A 70-years-old woman with a 13-years history of Parkinsons disease (PD) was hospitalized due to difficulty walking, despite being treated with levodopa/carbidopa (600 mg/day), immediate-release pramipexole (3 mg/day), and selegiline (5 mg/day). Immediate-release pramipexole was changed to extended-release pramipexole without changing the dose or levodopa equivalent dose (LED). The patients adherence to drugs was good. The parkinsonism gradually improved and the patient was discharged. One month later, the patient developed severe generalized athetotic dyskinesia with visual hallucinations and hyperpyrexia that lasted for a week, and she was readmitted to hospital. On admission, the patient was conscious but slightly disoriented. Body temperature was 40.3°C with hyperhidrosis. Leukocyte count in the peripheral blood was 1.78×10(4)/ml and serum creatine kinase was >3×10(4) U/l. Chest survey, whole-body computed tomography, and cranial magnetic resonance imaging showed no abnormalities. The patient was diagnosed with DHS and treated by tapering the oral administration of dopaminergic drugs, including extended-release pramipexole. Her clinical condition recovered without dyskinesia, and serum creatine kinase level swiftly normalized. DHS and resemblant conditions are reported to occur in long-term PD patients with motor complications. In advanced stage PD, loss of dopaminergic neurons impairs the dopamine holding capacity of the striatum and exogenous dopaminergic drugs can result in uncontrollable and excessive fluctuations in dopamine concentration. Our case recommends caution when switching to long-acting dopaminergic drugs, even if the dose is unchanged, could lead to excessive dopaminergic stimulation. This case highlights the importance of considering both the LED and the duration of action of dopaminergic drugs when adjusting medication.