Macroglossia in advanced amyotrophic lateral sclerosis.

INTRODUCTION: An enlarged tongue (macroglossia) has been reported in advanced-stage patients with amyotrophic lateral sclerosis (ALS). METHODS: In this study we examined the prevalence of macroglossia and analyzed clinical correlations in 65 ALS patients on tracheostomy-invasive ventilation (TIV). RESULTS: Macroglossia was found in 22 patients (33.8%). Compared with those without macroglossia, patients with macroglossia had a younger age of onset, longer duration of disease and TIV use, lower ALS Functional Rating Scale score, higher body mass index, lower energy intake, more severe communication impairment, and lower oral function. Logistic multivariate analysis showed that body mass index (BMI; P = 0.007) and communication impairment (P = 0.029) were significantly correlated with macroglossia. The duration of TIV use was at the cut-off level of significance (P = 0.05). CONCLUSIONS: Macroglossia may be the result of overfeeding and replacement by fat during long-term TIV use in patients with advanced ALS. Muscle Nerve, 2016 Muscle Nerve 54: 386-390, 2016.

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.

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.

Inhibition of cyclooxygenase-1 by nonsteroidal anti-inflammatory drugs demethylates MeR2 enhancer and promotes Mbnl1 transcription in myogenic cells.

Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA-binding protein, which is highly expressed in skeletal muscle. Abnormally expanded CUG-repeats in the DMPK gene cause myotonic dystrophy type 1 (DM1) by sequestration of MBNL1 to nuclear RNA foci and by upregulation of another RNA-binding protein, CUG-binding protein 1 (CUGBP1). We previously reported that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone, upregulates MBNL1 expression in DM1 mouse model by demethylation of MeR2, an enhancer element in Mbnl1 intron 1. NSAIDs inhibit cyclooxygenase (COX), which is comprised of COX-1 and COX-2 isoforms. In this study, we screened 29 NSAIDs in C2C12 myoblasts, and found that 13 NSAIDs enhanced Mbnl1 expression, where COX-1-selective NSAIDs upregulated Mbnl1 more than COX-2-selective NSAIDs. Consistently, knockdown of COX-1, but not of COX-2, upregulated MBNL1 expression in C2C12 myoblasts and myotubes, as well as in myotubes differentiated from DM1 patient-derived induced pluripotent stem cells (iPSCs). Luciferase assay showed that COX-1-knockdown augmented the MeR2 enhancer activity. Furthermore, bisulfite sequencing analysis demonstrated that COX-1-knockdown suppressed methylation of MeR2. These results suggest that COX-1 inhibition upregulates Mbnl1 transcription through demethylation of the MeR2 enhancer. Taken together, our study provides new insights into the transcriptional regulation of Mbnl1 by the COX-1-mediated pathway.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus.

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus ). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3'-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Analysis of resting salivation rate in patients with amyotrophic lateral sclerosis using tracheostomy invasive ventilation.

Patients with amyotrophic lateral sclerosis (ALS) often suffer from salivation problems such as drooling and dry mouth. We examined resting salivation rate cross-sectionally in 66 advanced ALS patients with tracheostomy invasive ventilation using a cotton roll method, and investigated clinical factors associated with salivation rate. Resting salivation rate in the patients was well preserved (median value 0.6 g/min), and was significantly more increased in patients with impairment of jaw movement (P = 0.007) or mouth opening (P = 0.003) than in patients with less impairment, and in patients with the mouth being constantly open ≥ 10 mm in rostrocaudal length than in patients with < 10 mm. These data indicate that salivation rate was increased with progression of dysfunction of voluntary jaw movement. Appropriate oral care is required in advanced ALS patients to maintain their oral hygiene and to avoid penetration of saliva into the airway.

Phenylbutazone induces expression of MBNL1 and suppresses formation of MBNL1-CUG RNA foci in a mouse model of myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is caused by abnormal expansion of CTG repeats in the 3' untranslated region of the DMPK gene. Expanded CTG repeats are transcribed into RNA and make an aggregate with a splicing regulator, MBNL1, in the nucleus, which is called the nuclear foci. The nuclear foci sequestrates and downregulates availability of MBNL1. Symptomatic treatments are available for DM1, but no rational therapy is available. In this study, we found that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone (PBZ), upregulated the expression of MBNL1 in C2C12 myoblasts as well as in the HSA(LR) mouse model for DM1. In the DM1 mice model, PBZ ameliorated aberrant splicing of Clcn1, Nfix, and Rpn2. PBZ increased expression of skeletal muscle chloride channel, decreased abnormal central nuclei of muscle fibers, and improved wheel-running activity in HSA(LR) mice. We found that the effect of PBZ was conferred by two distinct mechanisms. First, PBZ suppressed methylation of an enhancer region in Mbnl1 intron 1, and enhanced transcription of Mbnl1 mRNA. Second, PBZ attenuated binding of MBNL1 to abnormally expanded CUG repeats in cellulo and in vitro. Our studies suggest that PBZ is a potent therapeutic agent for DM1 that upregulates availability of MBNL1.

A patient with myotonic dystrophy type 1 (DM 1) accompanied by laryngeal and renal cell carcinomas had a small CTG triplet repeat expansion but no somatic instability in normal tissues.

We examined (CTG)n lengths in various tissues from a 70-year-old man with myotonic dystrophy type 1 (DM 1) who had a small 60-70 (CTG), expansion in his leukocytes. He died of renal cell carcinoma 5 years after a total laryngectomy for laryngeal carcinoma. Southern blot and polymerase chain reaction analyses were done on tissues obtained at autopsy. In the various normal tissues, (CTG). lengths were almost all the same size, whereas the renal cell carcinoma and metastatic tissues had longer lengths. When compared with the lengths in leukocytes about 5 years previously, (CTG)n lengths in the normal tissues were the same size. These findings suggest that both somatic instability and age-dependent (CTG). expansion in DM 1 patients with a small expansion may be less dominant than in patients with large expansions.

Thr1313Met mutation in skeletal muscle sodium channels in a Japanese family with paramyotonia congenita.

A 37-year-old Japanese woman was referred from another clinic to confirm the diagnosis of myotonia congenita. She had experienced cold-induced myotonia and muscle stiffness from early childhood. Of her three children, her elder son and her daughter have clinical features similar to hers. They experience neither grip nor percussion myotonia during warm weather, whereas myotonia is provoked by cold. Her younger son has no symptoms. DNA analyses of the SCN4A gene showed a C to T transition at nucleotide position 3938 in exon 22 of SCN4A (Thr1313Met) in all three affected family members, but not in the unaffected son. Paramyotonia congenita, the prevalence of which is very low in Japan, was diagnosed based on their clinical features and DNA analysis results.

A case of water intoxication with prolonged hyponatremia caused by excessive water drinking and secondary SIADH.

Water intoxication is a life-threatening disorder accompanied by brain function impairment due to severe dilutional hyponatremia. We treated a 22-year-old man without psychotic illness who had been put in a detention facility. He drank 6 liters of water over a 3-hour period at the facility as a game's penalty, and he showed progressive psychiatric and neurological signs including restlessness, peculiar behavior and convulsions. On his admission, 15 h after the discontinuation of the water drinking, he was in a coma, showing intermittent convulsions and remarkable hyponatremia (120 mmol/l). Because his laboratory tests showed hypertonic urine and normal sodium excretion, the diagnosis of secondary development of syndrome of inappropriate secretion of antidiuretic hormone (SIADH) was strongly suggested and later confirmed by the suppression of the renin-aldosterone system and the inappropriately elevated secretion of ADH. Saline infusion and an initial administration of furosemide in addition to dexamethasone as treatments for the patient's brain edema successfully improved his laboratory data and clinical signs by the 3rd hospital day, and he was returned to the facility without physical or psychiatric abnormalities on the 6th day. The secondary SIADH might have been due to the prolonged emesis, recurrent convulsions and rapid elevation of intracranial pressure.

New mutation of the Na channel in the severe form of potassium-aggravated myotonia.

Myotonia manifests in several hereditary diseases, including hyperkalemic periodic paralysis (HyperPP), paramyotonia congenita (PMC), and potassium-aggravated myotonia (PAM). These are allelic disorders originating from missense mutations in the gene that codes the skeletal muscle sodium channel, Nav1.4. Moreover, a severe form of PAM has been designated as myotonia permanens. A new mutation of Nav1.4, Q1633E, was identified in a Japanese family presenting with the PAM phenotype. The proband suffered from cyanotic attacks during infancy. The mutated amino acid residue is located on the EF-hand calcium-binding motif in the intracellular C-terminus. A functional analysis of the mutant channel using the voltage-clamp method revealed disruption of fast inactivation, a slower rate of current decay, and a depolarized shift in the voltage dependence of availability. This study has identified a new mutation of PAM with a severe phenotype and emphasizes the importance of the C-terminus for fast inactivation of the sodium channel. Muscle Nerve 39: 666-673, 2009.

Eye movement disorders in myotonic dystrophy type 1.

CONCLUSIONS: No definite sign was found of central oculomotor system disorders being independent of saccadic slowing because (1) diminished maximum slow phase velocity of the optokinetic nystagmus (OKNspv) was closely related to saccadic slowing (p<0.01, r=0.59), (2) maximum frequency of optokinetic nystagmus (OKNfq) was normal, (3) visual suppression (VS) change was mild, and (4) the diminished maximum slow phase velocity of the caloric nystagmus (CNspv) seen in some patients is explained by both peripheral and central vestibular involvement. These findings support the extraocular muscle hypothesis. OBJECTIVE: To assess whether eye movement disorders seen in patients with myotonic dystrophy type 1 (DM1) are caused by central oculomotor system involvement or extraocular muscle damage. PATIENTS AND METHODS: Oculomotor functions and their correlation with (CTG)n length were studied in 29 DM1 patients and 12 age-matched controls. RESULTS: Values for saccadic velocity (p<0.005), maximum OKNspv (p<0.005), and maximum CNspv (p<0.01) in the patient group were markedly lower than in the control group. VS of caloric nystagmus in the patient group was slightly lower than that in the controls. No significant difference was found between the two groups in the maximum OKNfq. Patients with greater (CTG)n lengths had lower saccadic velocities (p<0.01, r=0.71).