Junko Nishioka

L-arginine improves the symptoms of strokelike episodes in MELAS.

Based on the hypothesis that mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) are caused by impaired vasodilation in an intracerebral artery, the authors evaluated the effects of administering l-arginine, a nitric oxide precursor. Patients were administered l-arginine intravenously at the acute phase or orally at the interictal phase. l-Arginine infusions significantly improved all strokelike symptoms, suggesting that oral administration within 30 minutes of a stroke significantly decreased frequency and severity of strokelike episodes.

MELAS and l-arginine therapy: pathophysiology of stroke-like episodes

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke‐like episodes (MELAS) is a maternally inherited multisystem mitochondrial disorder. Although many molecular and cellular mechanisms have been discovered leading to mitochondrial cytopathy, the pathogenic mechanism of stroke‐like episodes seen in MELAS has not been clarified yet. According to the muscle and brain pathology and vascular physiology, mitochondrial angiopathy, or endothelial dysfunction, were proposed to play an important role for developing stroke‐like episodes. Based on a hypothesis of mitochondrial angiopathy theory, we use l‐arginine in MELAS patients and report its usefulness. This review aims to give a general idea on the actual knowledge about the possible pathogenic mechanism of stroke‐like episodes, including clinical symptoms that lead to stroke‐like episodes, muscle, or brain pathology, molecular cellular functions, neuroimagings including MRI, MRS, and SPECT, and the proposed site of action of l‐arginine therapy on MELAS patients. Currently, l‐arginine therapy may be the most promising for the treatment of stroke‐like episodes in MELAS.

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.

Molecular pathology of MELAS and L-arginine effects.

BACKGROUND The pathogenic mechanism of stroke-like episodes seen in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) has not been clarified yet. About 80% of MELAS patients have an A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene, which is the base change at position 14 in the consensus structure of tRNA(Leu(UUR)) gene. SCOPE OF REVIEW This review aims to give an overview on the actual knowledge about the pathogenic mechanism of mitochondrial cytopathy at the molecular levels, the possible pathogenic mechanism of mitochondrial angiopathy to cause stroke-like episodes at the clinical and pathophysiological levels, and the proposed site of action of l-arginine therapy on MELAS. MAJOR CONCLUSIONS Molecular pathogenesis is mainly demonstrated using ρ(0) cybrid system. The mutation creates the protein synthesis defects caused by 1) decreased life span of steady state amount of tRNA(Leu(UUR)) molecules; 2) decreased ratio of aminoacyl-tRNA(Leu(UUR)) versus uncharged tRNA(Leu(UUR)) molecules; 3) the accumulation of aminoacylation with leucine without any misacylation; 4) accumulation of processing intermediates such as RNA 19, 5) wobble modification defects. All of these loss of function abnormalities are created by the threshold effects of cell or organ to the mitochondrial energy requirement when they establish the phenotype. Mitochondrial angiopathy demonstrated by muscle or brain pathology, as SSV (SDH strongly stained vessels), and by vascular physiology using FMD (flow mediated dilation). MELAS patients show decreased capacity of NO dependent vasodilation because of the low plasma levels of l-arginine and/or of respiratory chain dysfunction. Although the underlying mechanisms are not completely understood in stroke-like episodes in MELAS, l-arginine therapy improved endothelial dysfunction. GENERAL SIGNIFICANCE Though the molecular pathogenesis of an A3243G or T3271C mutation of mitochondrial tRNA(Leu(UUR)) gene has been clarified as a mitochondrial cytopathy, the underlying mechanisms of stroke-like episodes in MELAS are not completely understood. At this point, l-arginine therapy showed promise in treating of the stroke-like episodes in MELAS. This article is part of a Special Issue entitled Biochemistry of Mitochondria.

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.

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.

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.

Molecular and clinical analyses of two patients with UPD(16)mat detected by screening 94 patients with Silver-Russell syndrome phenotype of unknown aetiology

Background Recently, a patient with maternal uniparental disomy of chromosome 16 (UPD(16)mat) presenting with Silver-Russell syndrome (SRS) phenotype was reported. SRS is characterised by growth failure and dysmorphic features. Objective To clarify the prevalence of UPD(16)mat in aetiology-unknown patients with SRS phenotype and phenotypic differences between UPD(16)mat and SRS. Methods We studied 94 patients with SRS phenotype of unknown aetiology. Sixty-three satisfied the Netchine-Harbison clinical scoring system (NH-CSS) criteria, and 25 out of 63 patients showed both protruding forehead and relative macrocephaly (clinical SRS). The remaining 31 patients met only three NH-CSS criteria, but were clinically suspected as having SRS. To detect UPD(16)mat, we performed methylation analysis for the ZNF597:TSS-differentially methylated region (DMR) on chromosome 16 and subsequently performed microsatellite, SNP array and exome analyses in the patients with hypomethylated ZNF597:TSS-DMR. Results We identified two patients (2.1%) with a mixture of maternal isodisomy and heterodisomy of chromosome 16 in 94 aetiology-unknown patients with SRS phenotype. Both patients exhibited preterm birth and prenatal and postnatal growth failure. The male patient had ventricular septal defect and hypospadias. Whole-exome sequencing detected no gene mutations related to their phenotypes. Conclusion We suggest considering genetic testing for UPD(16)mat in SRS phenotypic patients without known aetiology.

The first Japanese case of central precocious puberty with a novel MKRN3 mutation

MKRN3, located on chromosome 15q11.2, encodes makorin ring-finger 3, which is an upstream suppressor of the hypothalamic-pituitary-gonadal axis. Mutation of this gene induces central precocious puberty (CPP). As MKRN3 is maternally imprinted, only the paternal allele is expressed. This is the first report of an 8-year-old Japanese girl with CPP caused by a novel frameshift mutation in MKRN3 (p.Glu229Argfs*3).

A two-day-old hyperthyroid neonate with thyroid hormone resistance born to a mother with well-controlled Graves’ disease: a case report

IntroductionResistance to thyroid hormone is a syndrome caused by thyroid hormone receptor β mutations, which are usually inherited in an autosomal-dominant pattern.Case presentationOur patient, a Japanese neonate boy, showed hyperthyroid symptoms at age two days. Although our patient was diagnosed as having resistance to thyroid hormone, his hyperthyroid symptoms continued for two weeks. Therefore, our patient was treated with methimazole and iodine for two weeks from birth, showing no side effects and no symptoms upon treatment. At age 70 days, an R243W mutation in thyroid hormone receptor β was detected in our patient; while absent in his mother, the mutation was present in his father, who never showed any symptoms.ConclusionsTo the best of our knowledge this is the first case report of a resistance to thyroid hormone in a neonate presenting with hyperthyroid symptoms born to a mother with Graves’ disease and treated with methimazole and iodine. These results suggest that methimazole and iodine may be a good short-term option for treatment.