Novel mitochondrial DNA ND5 mutation in a patient with clinical features of MELAS and MERRF

BACKGROUND: The mitochondrial DNA gene encoding subunit 5 of complex I (ND5) has turned out to be a hot spot for mutations associated with mitochondrial encephalomyopathy with lactic acidosis and strokelike episodes (MELAS) and various overlap syndromes. OBJECTIVE: To describe a novel mutation in the ND5 gene in a young man man with an overlap syndrome of MELAS and myoclonus epilepsy with ragged-red fibers. DESIGN: Case report. PATIENT: A 25-year-old man had recurrent strokes, seizures, and myoclonus. His mother also had multiple strokes. A muscle biopsy specimen showed no ragged-red fibers but several strongly succinate dehydrogenase-reactive blood vessels. RESULTS: Biochemical analysis showed isolated complex I deficiency and molecular analysis revealed a novel heteroplasmic mutation (G13042A) in the ND5 gene. CONCLUSIONS: These data confirm that ND5 is a genetic hot spot for overlap syndromes, including MELAS and strokelike and myoclonus epilepsy with ragged-red fibers.     

A novel mtDNA mutation in the ND5 subunit of complex I in two MELAS patients

We identified a novel heteroplasmic mutation in the mitochodrial DNA gene encoding the ND5 subunit of complex I. This mutation (13514A-->G) hits the same codon affected by a previously reported mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS)-associated mutation (13513G-->A), but the amino acid replacement is different (D393G vs D393N). The 13514A-->G mutation was found in two unrelated MELAS-like patients. However, in contrast to typical MELAS, lactic acidosis was absent or mild and the muscle biopsy was morphologically normal. Strongly positive correlation between the percentage of heteroplasmy and defective activity of complex I was found in cybrids. We found an additional 13513G-->A-positive case, affected by a progressive mitochondrial encephalomyopathy. Our results clearly demonstrate that the amino acid position D393 is crucial for the function of complex I. Search for D393 mutations should be part of the routine screening for mitochondrial disorders.     

Is the mitochondrial complex I ND5 gene a hot‐spot for MELAS causing mutations?

We identified two novel heteroplasmic mitochondrial DNA point mutations in the gene encoding the ND5 subunit of complex I: a 12770A-->G transition identified in a patient with MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and a 13045A-->C transversion in a patient with a MELAS/Leber's hereditary optic neuropathy/Leigh's overlap syndrome. Biochemical analysis of muscle homogenates showed normal or very mildly reduced complex I activity. Histochemistry was normal. Our observations add to the evidence that mitochondrial ND5 protein coding gene mutations frequently associate with the MELAS phenotype, and it highlights the role of complex I dysfunction in MELAS.     

The G13513A mutation in the ND5 gene of mitochondrial DNA as a common cause of MELAS or Leigh syndrome: evidence from 12 cases

BACKGROUND: The number of molecular causes of MELAS (a syndrome consisting of mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes) and Leigh syndrome (LS) has steadily increased. Among these, mutations in the ND5 gene (OMIM 516005) of mitochondrial DNA are important, and the A13513A change has emerged as a hotspot. OBJECTIVE: To describe the clinical features, muscle pathological and biochemical characteristics, and molecular study findings of 12 patients harboring the G13513A mutation in the ND5 gene of mitochondrial DNA compared with 14 previously described patients with the same mutation. DESIGN: Clinical examinations and morphological, biochemical, and molecular analyses. SETTING: Tertiary care university hospital and molecular diagnostic laboratory. PATIENTS: Three patients had the typical syndrome features of MELAS; the other 9 had typical clinical and radiological features of LS. RESULTS: Family history suggested maternal inheritance in a few cases; morphological studies of muscle samples rarely showed typical ragged-red fibers and more often exhibited strongly succinate dehydrogenase-reactive blood vessels. Biochemically, complex I deficiency was inconsistent and generally mild. The mutation load was relatively high in the muscle and blood specimens. CONCLUSION: The G13513A mutation is a common cause of MELAS and LS, even in the absence of obvious maternal inheritance, pathological findings in muscle, or severe complex I deficiency.     

De novo mutations in the mitochondrial ND3 gene as a cause of infantile mitochondrial encephalopathy and complex I deficiency

Both nuclear and mitochondrial DNA mutations can cause energy generation disorders. Respiratory chain complex I deficiency is the most common energy generation disorder and a frequent cause of infantile mitochondrial encephalopathies such as Leigh's disease and lethal infantile mitochondrial disease. Most such cases have been assumed to be caused by nuclear gene defects, but recently an increasing number have been shown to be caused by mutations in the mitochondrially encoded complex I subunit genes ND4, ND5, and ND6. We report the first four cases of infantile mitochondrial encephalopathies caused by mutations in the ND3 subunit gene. Three unrelated children have the same novel heteroplasmic mutation (T10158C), only the second mutation reported in ND3, and one has the previously identified T10191C mutation. Both mutations cause disproportionately greater reductions in enzyme activity than in the amount of fully assembled complex I, suggesting the ND3 subunit plays an unknown but important role in electron transport, proton pumping, or ubiquinone binding. Three cases appear to have a de novo mutation, with no mutation detected in maternal relatives. Mitochondrial DNA disease may be considerably more prevalent in the pediatric population than currently predicted and should be considered in patients with infantile mitochondrial encephalopathies and complex I deficiency.     

A novel tRNA(Val) mitochondrial DNA mutation causing MELAS

Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is the most common mitochondrial disease due to mitochondrial DNA (mtDNA) mutations. At least 15 distinct mtDNA mutations have been associated with MELAS, and about 80% of the cases are caused by the A3243G tRNA(Leu(UUR)) gene mutation. We report here a novel tRNA(Val) mutation in a 37-year-old woman with manifestations of MELAS, and compare her clinicopathological phenotype with other rare cases associated tRNA(Val) mutations.     

Transition from Leigh syndrome to MELAS syndrome in a patient with heteroplasmic MT-ND3 m.10158T>C

An m.10158T>C mutation in MT-ND3, encoding a subunit of respiratory complex I, causes early-onset Leigh syndrome (LS), mitochondrial encephalomyopathy with lactic acid and stroke-like episodes (MELAS) syndrome, and LS and MELAS overlapping syndrome, presumably dependent on the ratio of heteroplasmy. Herein, we report a 4-year-old girl with heteroplasmic m.10158T>C mutation, showing an evolving age-dependent phenotype from LS to MELAS syndromes. She showed mild developmental delay during infancy, which was associated with magnetic resonance imaging lesions in the brain stem and basal ganglia. At the age of 4 years, she developed rapid neurological deterioration and intractable seizures, which was associated with recurrent multiple cerebral lesions as well as basal ganglia lesions. Her cerebral lesions were located predominantly in white matter and appeared at multiple areas simultaneously, unique characteristics that are distinct from typical MELAS. Two patients with LS-MELAS overlapping syndrome with m.10158T>C have been previously reported, however, this is the first patient with m.10158T>C showing significant age-dependent changes in clinical features and neuro-images, implying an age-dependent role of complex I in the developing brain.     

A novel nonsense variant in MT-CO3 causes MELAS syndrome

Both mitochondrial and nuclear gene mutations can cause cytochrome c oxidase (COX, complex Ⅳ) dysfunction, leading to mitochondrial diseases. Although numerous diseases caused by defects of the COX subunits or COX assembly factors have been documented, clinical cases directly related to mitochondrial cytochrome c oxidase subunit 3 gene (MT-CO3) mutations are relatively rare. Here, we report a 47-year-old female patient presented with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Muscle pathology revealed ragged-red fibres and remarkable COX-deficient muscle fibres. Muscle mitochondrial DNA sequencing analysis identified a novel MT-CO3 variant (m.9553G>A) that changed a highly conserved amino acid to a stop codon (p.Trp116*). This variant was heteroplasmic in multiple tissues, where the mutation load was 13% in oral epithelial cells, 89% in muscle samples, and not detectable in the peripheral blood lymphocytes. Single muscle fiber PCR analysis showed clear segregation of the mutation load with COX deficient fibres. Western blot analysis of the muscle samples revealed a significant decrease in the levels of COX1, COX2, COX3, COX4 and UQCRC2. COX respiration activity was remarkably reduced (58.84%) relative to the controls according to spectrophotometric assays. Taken together, our results indicated that this m.9553G>A variant may be responsible for the MELAS symdrome in the proband by affecting the stability and function of COX. The study expands the clinical and molecular spectrum of COX3-specific mitochondrial diseases.     

A missense mutation in the mitochondrial ND5 gene associated with a Leigh-MELAS overlap syndrome

A 13084 A->T missense mutation in the mitochondrial ND5 gene was identified in a 16-year-old boy affected with a progressive neurodegenerative disorder combining features of Leigh and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes) syndromes. Muscle biopsy analysis revealed partial complex I deficiency. The mutation presented a variable degree of heteroplasmy in the patient's tissues. This finding underlines the contribution of mtDNA-encoded complex I subunits in the etiology of complex I deficiency associated with encephalopathy.     

A MELAS-associated ND1 mutation causing leber hereditary optic neuropathy and spastic dystonia

OBJECTIVE: To report a novel mutation that is associated with Leber hereditary optic neuropathy (LHON) within the same family affected by spastic dystonia. DESIGN: Leber hereditary optic neuropathy is a mitochondrial disorder characterized by isolated central visual loss. Of patients with LHON, 95% carry a mutation in 1 of 3 mitochondrial DNA-encoded complex I genes. The complete mitochondrial DNA was screened for mutations in a patient with LHON without 1 of these 3 primary mutations. The heteroplasmy level and biochemical consequence of the mutation were determined. RESULTS: A pathogenic 3697G>A/ND1 mutation was detected and seemed associated with an isolated complex I deficiency. This family has similar clinical characteristics as the previously described families with LHON and dystonia with an ND6 mutation. CONCLUSIONS: The 3697G>A/ND1 mitochondrial DNA mutation causes the LHON and spastic dystonia phenotype in the same family. This mutation can also cause MELAS syndrome (which encompasses mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke), and other genetic factors may contribute to the clinical expression.     

MELAS: clinical features, biochemistry, and molecular genetics.

We studied 23 patients with clinically defined mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), 25 oligosymptomatic or asymptomatic maternal relatives, and 50 mitochondrial disease control subjects for the presence of a previously reported heteroplasmic point mutation at nt 3,243 in the transfer RNA(Leu(UUR)) gene of mitochondrial DNA. We found a high concordance between clinical diagnosis of MELAS and transfer RNA(Leu(UUR)) mutation, which was present in 21 of the 23 patients with MELAS, all 11 oligosymptomatic and 12 of 14 asymptomatic relatives, but in only five of 50 patients without MELAS. The proportion of mutant genomes in muscle ranged from 56 to 95% and was significantly higher in the patients with MELAS than in their oligosymptomatic or asymptomatic relatives. In subjects in whom both muscle and blood were studied, the percentage of mutations was significantly lower in blood and was not detected in three of 12 asymptomatic relatives. The activities of complexes I + III, II + III, and IV were decreased in muscle biopsies harboring the mutation, but there was no clear correlation between percentage of mutant mitochondrial DNAs and severity of the biochemical defect.     

A 4–base pair deletion in the mitochondrial cytochrome b gene associated with parkinsonism/MELAS overlap syndrome

Five patients with diminished activity of complex III of the mitochondrial respiratory chain have been screened for mutations in the mitochondrial cytochrome b (cyt b) gene. In 1 patient, a young boy with an akinetic rigid syndrome and a mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), a novel 4–base pair deletion was identified. This mutation in this highly conserved gene is considered to be pathogenic since it is a heteroplasmic frame shift mutation predicted to lead to a truncated protein. Ann Neurol 1999;45:130–133     

线粒体脑肌病伴有高乳酸血症和卒中样发作综合征的临床及基因突变分析

目的 探讨线粒体脑肌病伴有高乳酸血症和卒中样发作综合征(MELAS)的临床及基因突变特征. 方法 对1例MELAS患者的临床表现、影像学、肌肉病理特点进行分析,并用PCR-RFLP结合基因测序方法进行线粒体基因突变分析. 结果 患者主要临床表现为发作性头痛和呕吐、反复卒中样发作、癫痫、运动不耐受、身材矮小、神经性耳聋、乳酸水平升高等.脑CT见双侧基底节多个钙化灶,MRI见枕叶异常信号,1H-MRS见T2WI异常信号区域有明显的乳酸峰,在T2正常信号区域也有小的乳酸峰.光镜及电镜肌肉病理检查未见明显的线粒体异常,基因检测显示mtDNA A3243G杂合突变. 结论 MELAS的诊断必须结合临床表现、影像学、病理学和基因突变检测等结果进行综合分析,病理学检查阴性不能否定MELAS的诊断,诊断MELAS应常规进行mtDNA突变分析.     

Genetic, pathogenetic, and phenotypic implications of the mitochondrial A3243G tRNALeu(UUR) mutation

Mitochondrial disorders are frequently caused by mutations in mitochondrial genes and usually present as multisystem disease. One of the most frequent mitochondrial mutations is the A3,243G transition in the tRNALeu(UUR) gene. The phenotypic expression of the mutation is variable and comprises syndromic or non-syndromic mitochondrial disorders. Among the syndromic manifestations the mitochondrial encephalopathy, lactacidosis, and stroke-like episode (MELAS) syndrome is the most frequent. In single cases the A3,243G mutation may be associated with maternally inherited diabetes and deafness syndrome, myoclonic epilepsy and ragged-red fibers (MERRF) syndrome, MELAS/MERRF overlap syndrome, maternally inherited Leigh syndrome, chronic external ophthalmoplegia, or Kearns-Sayre syndrome. The wide phenotypic variability of the mutation is explained by the peculiarities of the mitochondrial DNA, such as heteroplasmy and mitotic segregation, resulting in different mutation loads in different tissues and family members. Moreover, there is some evidence that additional mtDNA sequence variations (polymorphisms, haplotypes) influence the phenotype of the A3,243G mutation. This review aims to give an overview on the actual knowledge about the genetic, pathogenetic, and phenotypic implications of the A3,243G mtDNA mutation.     

The mitochondrial DNA G13513A transition in ND5 is associated with a LHON/MELAS overlap syndrome and may be a frequent cause of MELAS

We report on 4 male patients with clinical, radiological, and muscle biopsy findings typical of the mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) phenotype. Skeletal muscle mitochondrial DNA (mtDNA) analysis showed that all patients harbored a heteroplasmic G13513A mutation in the ND5 subunit gene. One of these cases (Patient 1) presented with symptoms characteristic of Leber's hereditary optic neuropathy (LHON) 2 years before the first stroke-like episode. Quantitative analysis in several postmortem tissue sections showed that the relative proportions of mutant mtDNA were generally lower than those reported with other pathogenic mtDNA mutations. Single-fiber polymerase chain reaction studies demonstrated significantly higher amounts of mutant mtDNA in ragged red fibers (RRFs) compared with non-RRFs. This study indicates that the G13513A transition is likely to be pathogenic, that it can cause an LHON/MELAS overlap syndrome, and that it may be a more frequent cause of MELAS than previously recognized.     

A novel mitochondrial ND5 (MTND5) gene mutation giving isolated exercise intolerance

We describe a patient with isolated exercise intolerance caused by a new, maternally inherited mutation in mitochondrial DNA. The heteroplasmic T>C transition at position 13271 in MTND5 affects a highly conserved base and segregates with the disease, being present at highest levels in skeletal muscle fibres showing abnormal mitochondrial accumulation. This is the 15th mutation affecting the MTND5 subunit of respiratory chain complex I and confirms this protein as an important site for disease with phenotypes ranging from MELAS and infantile encephalopathies to isolated syndromes affecting a single tissue such as Leber hereditary optic neuropathy and now skeletal muscle.     

Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes correlates with heteroplasmic mutations of mitochondrial DNA 3243 A single-case genealogy analysis

BACKGROUND: Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) is a common syndrome of mitochondrial diseases caused primarily by a mutation from adenine to guanine at mitochondrial DNA 3243. However, the correlation between heteroplasmic mutations and clinical characteristics of hereditary MELAS syndrome is unclear. OBJECTIVE: To survey the clinical behaviors, biochemical outcomes, and imaging data in a patient with suspected MELAS syndrome by maternal inheritance, and to investigate the correlation with heteroplasmic mutations of hemocyte mitochondrial DNA. DESIGN, TIME AND SETTING: A case analysis based on hereditary family surgery was performed in the Enliang Hospital of Anshan, Taian County, and biochemical tests and gene diagnosis were erformed at the Department of Laboratory and Institute of Neurology, the First Affiliated Hospital of China Medical University, between March and September 2009. ARTICIPANTS: A 22-year-old female patient with MELAS syndrome was diagnosed in the First Affiliated Hospital of China Medical University in January, 2009. She had five males and seven females in her maternal family. METHODS: We obtained stroke and convulsion history in the patient and her family, as well as erforming routine blood tests, plasma lactic acid levels before and after movement, and magnetic resonance of the head. A mutation at m.3243A > G was verified using polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing, and quantitated using real-time polymerase chain reaction. MAIN OUTCOME MEASURES: Correlation of clinical behaviors and biochemical outcomes, as well as imaging data with heteroplasmic mutations in family members with typical and atypical MELAS syndrome. RESULTS: Some family members had typical symptoms of convulsion, stroke, and MELAS syndrome, as well as atypical symptoms of microsomia, movement intolerance, febrile, and migraine. Magnetic resonance of the head was consistent with typical imaging data of MELAS syndrome during attacks, and family members showed cerebellar atrophy. A heteroplasmic mutation of mitochondrial DNA 3243 occurred in all family members, although higher levels caused severe typical symptoms. The age of first-onset convulsion was negatively correlated with level of heteroplasmic mutation (r= -0.852, P< 0.05), but lactic acid was positively correlated with mutation levels (before movement, r= 0.945, P< 0.001; after movement, r= 0.945, P< 0.001). CONCLUSION: MELAS syndrome was diagnosed in this family by maternal inheritance, and the etiological factor was a mutation of mitochondrial A3243G. The level of heteroplasmic mutation correlated with anticipated convulsion and lactic acid levels.     

In situ hybridization of muscle mitochondrial mRNA in mitochondrial myopathies

Summary To determine whether a mitochondrial mRNA deficiency exists in mitochondrial myopathies, muscle biopsies from a patient with chronic progressive external ophthalmoplegia (CPEO) and a patient with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) were studied using in situ hybridization. Histochemistry and immunohistochemistry were performed along with hybridization. Hybridization reactions were widely distributed over the sarcoplasm of all muscle fibers in the patient with MELAS. In the patient with CPEO, 80% of the fibers showed a marked decrease in density of autoradiographic grains. This marked decrease corresponded to the histochemical and immunohistochemical findings of a very weak staining of cytochromec oxidase (CCO). The isotope-labeled cDNA probe used in in situ hybridization in this study complements a part of subunit I of CCO and a part of subunit II of complex I in the mitochondrial gene. Our results suggest a defect in the mRNA in this CPEO patient.Supported in part by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education and by a grant (62-2-05) from the National Center of Neurology and Psychiatry (NCNP) of the Ministry of Health and Welfare, Japan