Simultaneous measurement of the size and methylation of chromosome 4qA-D4Z4 repeats in facioscapulohumeral muscular dystrophy by long-read sequencing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscular disorder characterized by asymmetric muscle wasting and weakness. FSHD can be subdivided into two types: FSHD1, caused by contraction of the D4Z4 repeat on chromosome 4q35, and FSHD2, caused by mild contraction of the D4Z4 repeat plus aberrant hypomethylation mediated by genetic variants in SMCHD1, DNMT3B, or LRIF1. Genetic diagnosis of FSHD is challenging because of the complex procedures required. METHODS: We applied Nanopore CRISPR/Cas9-targeted resequencing for the diagnosis of FSHD by simultaneous detection of D4Z4 repeat length and methylation status at nucleotide level in genetically-confirmed and suspected patients. RESULTS: We found significant hypomethylation of contracted 4q-D4Z4 repeats in FSHD1, and both 4q- and 10q-D4Z4 repeats in FSHD2. We also found that the hypomethylation in the contracted D4Z4 in FSHD1 is moderately correlated with patient phenotypes. CONCLUSIONS: Our method contributes to the development for the diagnosis of FSHD using Nanopore long-read sequencing. This finding might give insight into the mechanisms by which repeat contraction causes disease pathogenesis.

Induction of Genes Expressed in Endothelial Cells of the Corpus Callosum in the Chronic Cerebral Hypoperfusion Rat Model.

BACKGROUND: Cerebrovascular white matter lesions (WMLs) are associated with cognitive impairment in patients with subcortical vascular dementia. We performed a comprehensive gene expression analysis to elucidate genes associated with WML development in a chronic cerebral hypoperfusion rat model. METHODS: Brains of rats with bilateral carotid ligation (2VO, n = 10) and sham-operated rats (n = 5-10/group) were removed on days 1, 7, or 28 after surgery. Total RNA isolated from the corpus callosum was evaluated by microarray analysis and quantitative reverse transcription-polymerase chain reaction. RESULTS: On days 7 and 28, WMLs exhibited histologic changes. On day 7, 16 genes were differentially expressed between groups. mRNA levels of Ptprb, Kcnj8, Crispld2, Bcl6b, and Gja5 were differentially expressed in 2VO rats on day 7, but then returned to normal, whereas mRNA levels of Vwf and Trappc6a were upregulated after day 7. Immunohistochemistry showed that GJA5 and vWF were detected in endothelial cells, KCNJ8 in endothelial cells and astrocytes, CRISPLD2 in neurons and astrocytes, and TRAPPC6A in neurons. CONCLUSION: Our findings indicate novel genes that may be associated with WML development in the chronic cerebral hypoperfusion rat model, and suggest an important role of neurovascular dysfunction in the pathophysiology.

Production of free glutamate in milk requires the leucine transporter LAT1.

The concentration of free glutamate (Glu) in rat's milk is ∼10 times higher than that in plasma. Previous work has shown that mammary tissue actively transports circulatory leucine (Leu), which is transaminated to synthesize other amino acids such as Glu and aspartate (Asp). To investigate the molecular basis of Leu transport and its conversion into Glu in the mammary gland, we characterized the expression of Leu transporters and [(3)H]Leu uptake in rat mammary cells. Gene expression analysis indicated that mammary cells express two Leu transporters, LAT1 and LAT2, with LAT1 being more abundant than LAT2. This transport system is sodium independent and transports large neutral amino acids. The Leu transport system in isolated rat mammary cells could be specifically blocked by the LAT1 inhibitors 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH) and triiodothyronine (T3). In organ cultures, Glu secretion was markedly inhibited by these LAT1 inhibitors. Furthermore, the profiles of Leu uptake inhibition by amino acids in mammary cells were similar to those reported for LAT1. In vivo, concentrations of free Glu and Asp increased in milk by oral gavage with Leu at 6, 12, and 18 days of lactation. These results indicate that the main Leu transporter in mammary tissue is LAT1 and the transport of Leu is a limiting factor for the synthesis and release of Glu and Asp into milk. Our studies provide the bases for the molecular mechanism of Leu transport in mammary tissue by LAT1 and its active role on free Glu secretion in milk, which confer umami taste in suckling pups.

[A case of limbic encephalitis repeated aphasic status epilepticus with periodic lateralized epileptiform discharges].

We report a case of limbic encephalitis repeated aphasic status epilepticus with periodic lateralized epileptiform discharges (PLEDs). A 51-year-old man developed convulsions, psychiatric symptoms such as anxiety, phobia and ease of anger, and Wernicke's aphasia. Analysis of the cerebrospinal fluid (CSF) showed increase of leukocyte count (148/microl, mononuclear cells). Brain magnetic resonance imaging (MRI) showed hyperintensity lesions in the left medial temporal area and basal frontal area on T2-weighted and fluid-attenuated inversion recovery (FLAIR) images. The electroencephalography (EEG) showed PLEDs over the left hemisphere, occurring at intervals of 0.5-1 Hz. Although his limbic symptoms improved, Wernicke's aphasia occurred periodically with PLEDs appearance. After the administration of antiepileptic drugs, his language performance improved, and PLEDs were completely disappeared. We diagnosed him limbic encephalitis with non-convulsive repeated aphasic status epilepticus with periodic lateralized epileptiform discharges. Aphasic status epilepticus should be considered in the patients with limbic encephalitis, and careful evaluation of aphasia and EEG should be necessary to diagnose of aphasic status epilepticus.

[A case of laminopathy with the mutation of LMNA gene identified by the exome analysis of disease-related genes].

Laminopathy, caused by mutations in the LMNA gene, include a variety of diseases, such as Emery-Dreifuss muscular dystrophy. A Japanese woman developed progressive muscle weakness, muscle atrophy and joint contractures of upper and lower limbs after the age of two years old. She had restrictive respiratory dysfunction, and developed both supraventricular and ventricular arrhythmias after the fourth decade of life. At 55 years old, she had tracheostomy, required mechanical ventilation and was implanted with the implantable cardioverter defibrillator. The serum level of creatine kinase was within normal range. Electromyography showed polyphasic or large motor unit potentials and reduced interference pattern, while relatively normal recruitment. The exome analysis of disease-related genes revealed a heterozygous pathogenic variant c.1072G>A (p.E358K) in the LMNA gene, which contributed to the diagnosis of laminopathy.

Induction of GNE in myofibers after muscle injury.

OBJECTIVE: The aim of the present study was to clarify the expression of uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) protein and mRNA in damaged or regenerating myofibers. METHODS: We investigated the muscle expression pattern of GNE protein by immunohistochemistry using a murine model involving intramuscular injection of cardiotoxin (CTX), and the expression level of GNE mRNA by quantitative real-time polymerase chain reaction analysis of damaged or regenerating myofibers that had been collected directly from tissue sections using laser-capture microdissection. RESULTS: The expression of GNE protein was increased in severely damaged myofibers as well as in regenerating myofibers with central nuclei, both of which also showed an increase in the expression of GNE mRNA. In regenerating myofibers, immunoreactivity for GNE protein in nuclei relative to that in the cytoplasm was higher at 7 days than at 4 days after CTX injection. CONCLUSION: Our findings suggest that GNE expression is induced when myofibers are damaged or regenerating, and that GNE plays a role in muscle regeneration.

[Case of Parkinson disease with heat retention due to sweating dysfunction].

A 71-year-old man was diagnosed as Parkinson disease at age 59, and levodopa therapy was started. Eleven years after the beginning of treatment, he noticed high fever (38.0 degrees C-39.0 degrees C) in July, but hyperthermia spontaneously disappeared three months later. In early July of the following year, he was re-admitted to our hospital because of continuous high fever, despite no any inflammation. Neurological examination revealed flexion posture of trunk and limbs and short step gait. He also presented limb rigidity, akinesia, and resting tremor during off period. Routine laboratory examinations and radiological examinations showed no remarkable findings. Autonomic testing revealed orthostatic hypotension and anhidrosis below trunk and lower limbs. By controlling the room temperature at 26 degrees C, hyperthermia showed a marked decline. In despite of no reports found associations between heat retention and Parkinson disease, in this case we speculate hyperthermia was caused by heat retention.

Corticobasal degeneration presenting with progressive conduction aphasia.

We report the case of a woman with primary progressive aphasia (PPA) presenting with conduction aphasia. Neurological findings showed bilateral finger tremor and signe de poignet figé in her right hand. Memory, orientation, and activities of daily living were well preserved. Linguistic examination showed severe impairment in repetition, fluent spontaneous speech with phonemic paraphasia, and relatively well preserved comprehension. Limb-kinetic apraxia and parkinsonism were not observed during the course of her illness. T1-weighted magnetic resonance imaging revealed severe atrophy of the left temporal lobe and dilatation of the left Sylvian fissure. Neuropathological findings demonstrated the most severe atrophy in the left superior temporal gyrus and Gallyas-Braak-positive or phosphorylated tau-immunoreactive cytoskeletal structures, which were consistent with corticobasal degeneration (CBD). We speculate that the progressive conduction aphasia of our patient might have been caused by left temporal lobe impairment. We suggest that progressive conduction aphasia may be a feature of CBD presenting with PPA.

Electronic structure of six-coordinate iron(III) monoazaporphyrins.

The electronic structures of six-coordinate iron(III) octaethylmonoazaporphyrins, [Fe(MAzP)L 2] (+/-) ( 1), have been examined by means of (1)H NMR and EPR spectroscopy to reveal the effect of meso-nitrogen in the porphyrin ring. The complexes carrying axial ligands with strong field strengths such as 1-MeIm, DMAP, CN (-), and (t)BuNC adopt the low-spin state with the (d xy ) (2)(d xz , d yz ) (3) ground state in a wide temperature range where the (1)H NMR and EPR spectra are taken. In contrast, the complexes with much weaker axial ligands, such as 4-CNPy and 3,5-Cl 2Py, exhibit the spin transition from the mainly S = 3/2 at 298 K to the S = 1/2 with the (d xy ) (2)(d xz , d yz ) (3) ground state at 4 K. Only the THF complex has maintained the S = 3/2 throughout the temperature range examined. Thus, the electronic structures of 1 resemble those of the corresponding iron(III) octaethylporphyrins, [Fe(OEP)L 2] (+/-) ( 2). A couple of differences have been observed, however, in the electronic structures of 1 and 2. One of the differences is the electronic ground state in low-spin bis( (t)BuNC) complexes. While [Fe(OEP)( (t)BuNC) 2] (+) adopts the (d xz , d yz ) (4)(d xy ) (1) ground state, like most of the bis( (t)BuNC) complexes reported previously, [Fe(MAzP)( (t)BuNC) 2] (+) has shown the (d xy ) (2)(d xz , d yz ) (3) ground state. Another difference is the spin state of the bis(3,5-Cl 2Py) complexes. While [Fe(OEP)(3,5-Cl 2Py) 2] (+) has maintained the mixed S = 3/2 and 5/2 spin state from 298 to 4 K, [Fe(MAzP)(3,5-Cl 2Py) 2] (+) has shown the spin transition mentioned above. These differences have been ascribed to the narrower N4 cavity and the presence of lower-lying pi* orbital in MAzP as compared with OEP.

Portal-systemic shunt encephalopathy presenting with diffuse cerebral white matter lesion: an autopsy case.

We report herein an autopsy case of portal-systemic encephalopathy (PSE) presenting with diffuse tissue rarefaction in the cerebral deep white matter. Clinically, the patient showed recurrent episodes of unconsciousness, abnormal behavior and urinary incontinence, as well as flapping tremor. Cognitive impairment and peripheral neuropathy developed following recurrent episodes. Although conventional arterial portography revealed a small portal-systemic collateral vessel of a left gastro-renal venous shunt, abdominal CT and liver biopsy showed no evidence of liver cirrhosis and serum ammonia level showed a mild increase. T2-weighted MRI demonstrated symmetrical signal hyperintensities in the deep white matter. Neuropathological findings showed Alzheimer type II astrocytes in the deep layers of the cerebral cortices and severe tissue rarefaction with no or slight reactive astrocytosis in the subcortical and deep white matter. These white matter changes have been reported infrequently in patients with PSE. The present case suggests that chronic PSE without liver cirrhosis may develop diffuse white matter lesions.

[A case of mitochondrial disease with multiple mitochondrial DNA deletions suspected amyotrophic lateral sclerosis-frontotemporal dementia].

A 76-year-old woman showed a dramatic lowering of her tone of voice in October 2014, followed by muscle weakness of the left arm. The previous attending physician noticed remarkable left dominant frontotemporal lobe atrophy on cranial MRI. Her dysarthria, dysphagia and the muscle weakness of her extremities worsened, and a muscle biopsy revealed mitochondrial abnormality. The mitochondrial DNA from her muscle showed multiple deletions; the previous physician therefore diagnosed the patient with mitochondrial disease. The patient resembled amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD). No other cases of ALS-FTD with mitochondrial disease have been reported in Japan. We therefore consider the present case to be valuable.

Relationship between White Matter Lesions and Progression of Cognitive Decline in Alzheimer's Disease.

BACKGROUND: This study examined the relationship between baseline white matter lesions (WMLs) and the progression of cognitive decline in patients with late-onset Alzheimer's disease (AD). METHODS: Fifty-six patients with AD were included in the study (23 men, 33 women; mean age, 77.8 years). All subjects were treated with acetylcholinesterase inhibitors and followed up for approximately 1 year. The Mini-Mental State Examination (MMSE) score was assessed at least twice to evaluate the progressive cognitive impairment. All subjects underwent brain MRI at baseline and were divided into WMLs(-), mild WMLs(+), and moderate WMLs(+) groups based on WML severity. Changes in MMSE scores between baseline and follow-up were analyzed using the Wilcoxon signed-rank test. RESULTS: MMSE scores at baseline did not differ significantly among the three groups (p = 0.1658), whereas MMSE scores at the follow-up evaluation were significantly lower in the moderate WMLs(+) group than in the WMLs(-) group (p = 0.0257). The mean MMSE scores remained above baseline values during the approximately 1-year follow-up in the WMLs(-) group, whereas they were decreased in the mild and moderate WMLs(+) groups. Moreover, the frequency of improvement in patients from the WMLs(-) group tended to be higher than that in patients from the WMLs(+) groups. CONCLUSION: Baseline WMLs may be associated with the heterogeneous progression of cognitive decline in patients with AD.

Monofocal large inflammatory demyelinating lesion, mimicking brain glioma.

Here we report two cases of pathologically confirmed tumor-like demyelinating lesions. In comparison with common primary demyelinating diseases, our cases demonstrated atypical radiologic features, such as a large monofocal lesion with mild brain edema, and open ring-like or focal enhancement on magnetic resonance images, suggesting brain tumors. The clinical manifestations included focal neurologic signs due to the lesions, monophasic episodes without relapse over a long follow-up period, and efficacy of oral corticosteroid therapy. Histological analysis of brain biopsy specimens showed the inflammatory demyelination and preserved axons without tumor cells. The present cases suggest the importance of considering inflammatory demyelinating disease in the different diagnosis of monofocal tumor-like lesion.

Role of ubiquitin-proteasome proteolysis in muscle fiber destruction in experimental chloroquine-induced myopathy.

Previous studies have documented the presence of rimmed vacuoles, atrophic fibers, and increased lysosomal cathepsin activity in skeletal muscle from animal models of chloroquine-induced myopathy, suggesting that muscle fibers in this type of myopathy may be degraded via the lysosomal-proteolysis pathway. Given recent evidence of abnormal ubiquitin accumulation in rimmed vacuoles, in this study we chose to examine the significance of the ubiquitin-proteasome proteolytic system in the process of muscle fiber destruction in experimental chloroquine myopathy. Expression of ubiquitin, 26S proteasome proteins, and ubiquitin ligases, such as muscle-specific RING finger-1 (MuRF-1) and atrogin-1/muscle atrophy F-box protein (MAFbx), was analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Abnormal accumulation of rimmed vacuoles was observed only in chloroquine-treated denervated muscles. Ubiquitin and proteasome immunostaining, and ubiquitin, MuRF-1, and atrogin-1/MAFbx mRNAs were significantly increased in denervated soleus muscles from saline- and chloroquine-treated rats when compared with contralateral innervated muscles. Further, ubiquitin and ubiquitin ligase mRNA levels were higher in denervated muscles from chloroquine-treated rats when compared with saline-treated rats. These data demonstrate increased proteasomes and ubiquitin in denervated muscles from chloroquine-treated rats and suggest that the ubiquitin-proteasome proteolysis pathway as well as the lysosomal-proteolysis pathway mediate muscle fiber destruction in experimental chloroquine myopathy.

Skeletal muscle expression of clathrin and mannose 6-phosphate receptor in experimental chloroquine-induced myopathy.

Previous studies suggest that the muscle fiber lysosome system plays a central role in the increased formation of autophagosomes and autolysosomes that occurs in the context of chloroquine-induced myopathy. The goal of this study was to characterize the contribution of receptor-mediated intracellular transport, particularly the endosomal pathway, to the abnormal accumulation of vacuoles in experimental chloroquine myopathy. Expression of the mannose 6-phosphate receptor (M6PR) and clathrin were analyzed in innervated and denervated rat soleus muscles after treatment with either saline or chloroquine. Accumulation of vacuoles was observed only in chloroquine-treated denervated muscles. Further, clathrin immunostaining and M6PR messenger ribonucleic acid (mRNA) were significantly increased in denervated soleus muscle from saline- and chloroquine-treated rats compared to contralateral, innervated muscles. However, there was no difference in clathrin levels when comparing saline- and chloroquine-treated denervated muscles. These data suggest that chloroquine activates the transport of newly synthesized lysosomal enzymes from the secretory pathway via the trans-Golgi network of the Golgi apparatus (an endosomal pathway) as well as autophagosome formation (an autophagic process) in skeletal muscles. Vacuoles may subsequently accumulate secondary to abnormal formation or turnover of autolysosomes at or after fusion of autophagosomes with early endosomes.