Shuichi Yatsuga

Effects of L-arginine on the acute phase of strokes in three patients with MELAS.

The primary cause for strokelike episodes in young patients with MELAS (myopathy, encephalopathy, lactic acidosis, and strokelike episodes)—whether mitochondrial cytopathy, angiopathy, or both—remains controversial. Based on a hypothesis that strokelike episodes in MELAS are caused by segmental impairment of vasodilation in intracerebral arteries, we administered l-arginine to three patients with MELAS in the acute phase of stroke (within 1 hour of onset) and evaluated effects on clinical course, biochemical measurements, and functional cerebral hemodynamics according to 99mTc-ECD SPECT. ### Patients. Patient 1 was a 17-year-old woman referred to the hospital for periodic vomiting, hemiconvulsion, and short stature (below 2.7 SD). Patient 2 was an 18-year-old woman who was admitted to the hospital for generalized muscle weakness, periodic vomiting, hemiparesis, and short stature (below 1.5 SD). Patient 3 was a 15-year-old boy referred to our hospital for hemiblindness, hemiconvulsions, vomiting, and short stature (below 2.8 SD). All patients showed extensive calcification in the basal ganglia, lactic acidosis (3.8 to 5.6 mmol/L; normal range …

MELAS: A nationwide prospective cohort study of 96 patients in Japan

BACKGROUND MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) (OMIM 540000) is the most dominant subtype of mitochondrial myopathy. The aim of this study was to determine the prevalence, natural course, and severity of MELAS. METHODS A prospective cohort study of 96 Japanese patients with MELAS was followed between June 2003 and April 2008. Patients with MELAS were identified and enrolled based on questionnaires administered to neurologists in Japan. MELAS was defined using the Japanese diagnostic criteria for MELAS. Two follow-up questionnaires were administered to neurologists managing MELAS patients at an interval of 5years. RESULTS A prevalence of at least 0.58 (95% confidential interval (CI), 0.54-0.62)/100,000 was calculated for mitochondrial myopathy, whereas the prevalence of MELAS was 0.18 (95%CI, 0.02-0.34)/100,000 in the total population. MELAS patients were divided into two sub-groups: juvenile form and adult form. Stroke-like episodes, seizure and headache were the most frequent symptoms seen in both forms of MELAS. Short stature was significantly more frequent in the juvenile form, whereas hearing loss, cortical blindness and diabetes mellitus were significantly more frequent in the adult form. According to the Japanese mitochondrial disease rating scale, MELAS patients showed rapidly increasing scores (mean±standard deviation, 12.8±8.7) within 5years from onset of the disease. According to a Kaplan-Meier analysis, the juvenile form was associated with a higher risk of death than the adult form (hazard ratio, 3.29; 95%CI, 1.32-8.20; p=0.0105). CONCLUSIONS AND GENERAL SIGNIFICANCE We confirmed that MELAS shows a rapid degenerative progression within a 5-year interval and that this occurs in both the juvenile and the adult forms of MELAS and follows different natural courses. This article is part of a Special Issue entitled: Biochemistry of Mitochondria.

Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders

The diagnosis of mitochondrial disorders (MDs) is occasionally difficult because patients often present with solitary, or a combination of, symptoms caused by each organ insufficiency, which may be the result of respiratory chain enzyme deficiency. Growth differentiation factor 15 (GDF‐15) has been reported to be elevated in serum of patients with MDs. In this study, we investigated whether GDF‐15 is a more useful biomarker for MDs than several conventional biomarkers.

GDF15 is a novel biomarker to evaluate efficacy of pyruvate therapy for mitochondrial diseases

Pyruvate therapy is a promising approach for the treatment of mitochondrial diseases. To identify novel biomarkers for diagnosis and to evaluate therapeutic efficacy, we performed microarray analysis of 2SD cybrid cells harboring a MELAS-causing mutation and control cells treated with either lactate or pyruvate. We found that expression and secretion of growth differentiation factor 15 (GDF15) were increased in 2SD cells treated with lactate and that serum GDF15 levels were significantly higher in patients with mitochondrial diseases than in those with other diseases, suggesting that GDF15 could be a useful marker for diagnosis and evaluating the therapeutic efficacy of pyruvate.

Inappropriate intracranial hemodynamics in the natural course of MELAS

The abnormalities of intracranial hemodynamics associated with strokelike episodes in MELAS are variable depend on the time phase from the onset of strokelike episodes and on the progression of the dementia state. To clarify the regional cerebral blood flows (rCBF) in the natural course of MELAS is very important to understand the pathogenic mechanism of this disorder, either cytopathy, angiopathy or both. We analyzed the serial studies of brain statistical parametric mapping (SPM) 99 single photon emission computed tomography (SPECT) in 5 MELAS patients in maximum 10 years interval, who fulfilled the clinical, pathological and genetic criteria of MELAS, and have an A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene. SPM is a proven and effective method for the voxel-by-voxel analysis of functional images which show the advantage in its promise of fully automated neurophysiological imaging analysis throughout the whole brain using various statistical analyses. SPECT acquisition was initiated and was reconstructed by iterative algorithm and were processed and analyzed with SPM 99 for Windows software. Statistics were displayed as Z scores (threshold: P < 0.01). The inappropriate intracranial hemodynamics was found not only at the acute but at the interictal phase, and was getting worse as the disease progress. Hypoperfusion in the posterior cingulate cortex was always observed (corrected P < 0.01) in MELAS patients, which is the typical finding reported in Alzheimers disease. The inappropriate intracranial hemodynamics is a common feature and may be related with mitochondrial angiopathy in the natural course of MELAS.

Increased mitochondrial processing intermediates associated with three tRNALeu(UUR) gene mutations

Accumulation of RNA 19 has been associated with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes. We analyzed total RNA in muscle specimens from six patients who had one of three pathogenetic point mutations in the mitochondrial tRNA(Leu(UUR)) gene, including A3243G, T3271C, and T3303C. Mitochondrial processing intermediates were identified and quantitated by Northern blotting. The percentage of DNA with the mutation also was determined in each patient. The intermediate (RNA 19) was significantly increased in all patients. The proportion of mutation-carrying RNA in processing intermediates was always higher than in the DNA fraction, suggesting that these mutations may have dominant-negative effects on mitochondrial RNA processing events at the tRNA(Leu(UUR)) gene boundary.

Genome-wide copy number analysis and systematic mutation screening in 58 patients with hypogonadotropic hypogonadism

OBJECTIVE To clarify the molecular basis of hypogonadotropic hypogonadism (HH). DESIGN Genome-wide copy number analysis by array-based comparative genomic hybridization and systematic mutation screening of 29 known causative genes by next-generation sequencing, followed by in silico functional assessment and messenger RNA/DNA analyses of the mutants/variants. SETTING Research institute. PATIENT(S) Fifty-eight patients with isolated HH (IHH), combined pituitary hormone deficiency (CPHD), and syndromic HH. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Frequency and character of molecular abnormalities. RESULT(S) Pathogenic defects were identified in 14 patients with various types of HH, although oligogenicity was not evident in this patient group. As rare abnormalities, we identified a submicroscopic deletion involving FGFR1 and an SOX3 polyalanine deletion in patients with IHH, and a WDR11 splice site mutation in a patient with CPHD. No disease-associated polymorphism was detected in the 58 patients. CONCLUSION(S) The present study provides further evidence that mutations and deletions in the known causative genes play a relatively minor role in the etiology of HH and that submicroscopic rearrangements encompassing FGFR1 can lead to IHH as a sole recognizable clinical feature. Furthermore, the results indicate for the first time that polyalanine deletions in SOX3 and mutations in WDR11 constitute rare genetic causes of IHH and CPHD, respectively.

Effect of l-arginine on synaptosomal mitochondrial function

OBJECTIVE Specific aim of this study is to elucidate the direct effects of L-arginine on the synaptosomal neurotransmission related to the mitochondrial respiratory function. METHODS Using isolated endbrains from wild-type mice (ICR), crude synaptosome was analyzed for their concentration of gamma-aminobutyric acid (GABA) and glutamate (Glu) with/without addition of L-arginine. We analyzed the contents of releasing amino acids evoked by high potassium condition and uptake of them in three separated fractions (cytosol, vesicles, and intact mitochondria). The oxygen consumption was also measured by oxygen electrode. RESULTS The entire uptakes of GABA and Glu were inhibited by rotenone (about 30 nmol/mg protein) with dose-dependent manner and showed a plateau at about 70% of total uptake. L-arginine inhibited the uptake of Glu logarithmically, however it showed no change in uptake of GABA. The contents of GABA and Glu in synaptosome were decreased in the presence of L-arginine. L-arginine enhanced the respiration of state II by succinate on synaptosomal respiration, although the respiration of synaptosomal mitochondrial fraction and the respiratory chains enzyme activities were almost unaffected by L-arginine. In the presence of rotenone, L-arginine decreased the uptake of Glu without changing the uptake of GABA, increased the releasing of GABA, and may modulate the excitability of neuronal state on the cytosol, cytomembrane, and/or organelles except for mitochondria. CONCLUSIONS L-arginine may modulate excitation by neurotransmitters at nerve endings, in relation to potentiated respiratory metabolism of succinate in synaptosomes. Such effects might contribute to alleviation of stroke-like symptoms in MELAS.

Safety and efficacy of treatment with asfotase alfa in patients with hypophosphatasia: Results from a Japanese clinical trial

Hypophosphatasia (HPP) is a rare skeletal disease characterized by hypomineralization and low alkaline phosphatase activity. Asfotase alfa (AA) has been recently developed to treat HPP complications. This study evaluated its safety and efficacy in Japan.

New TRPM6 mutation and management of hypomagnesaemia with secondary hypocalcaemia

BACKGROUND TRPM6 gene mutation has been reported to cause hypomagnesemia with secondary hypocalcemia (HSH). However, the genotype-phenotype correlation for TRPM6 gene mutations has not been clarified. OBJECTIVE To elucidate the factors underlying the severe neurological complications in HSH and evaluate the potential association between the location of TRPM6 gene mutations and clinical data of HSH. METHODS A Japanese patient diagnosed with HSH at 10 weeks of age exhibited neurological damage and failed to thrive. Magnesium supplements were therefore started at 12 weeks of age. Mutational analysis of the TRPM6 gene was performed using a direct sequencing method to determine the position and type of mutation. Using the data of 29 HSH patients reported in the literature, linear regression analysis was also performed to examine the association between TRPM6 gene mutation location and HSH onset age, initial serum magnesium and calcium concentrations, and dose of oral magnesium. RESULTS A novel stop-codon homozygous mutation [c.4190 G>A] W1397X was identified in exon 26 of the patients TRPM6 gene. No statistical correlation was found between the location of mutations in the TRPM6 gene and the clinical data for 4 clinical indicators of HSH. CONCLUSIONS We identified the first Japanese HSH patient with a novel nonsense mutation in the TRPM6 gene. Regression analysis of mutation locations in the protein-coding region of TRPM6 and the reported clinical data for 4 clinical indicators of HSH in 30 HSH patients did not detect a genotype-phenotype correlation.