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.     

Phenotypic patterns of MELAS/LS overlap syndrome associated with m.13513G>A mutation,and neuropathological findings in one autopsy case

The 13513G>A mutation in the ND5 gene of mitochondrial DNA (mtDNA) is usually associated with mitochondrial encephalomyopathy with lactate acidosis and stroke‐like episodes (MELAS), or Leigh syndrome (LS). In this study, we describe three young Chinese patients with MELAS/LS overlap syndrome who carried the m.13513G>A mutation. Clinical and MRI features were characteristic of both MELAS and LS. Interestingly, the clinical presentation of this overlap syndrome could be variable depending on the degree of relative contribution of MELAS and LS, that is, MELAS as the initial presenting syndrome, LS as the predominant syndrome, or both MELAS and LS appearing at the same time. The final brain MRI showed findings characteristic of both MELAS and LS, with asymmetrical lesions in the cortex and subcortical white matter of the occipital, temporal, and frontal lobes (MELAS), and bilateral and symmetrical lesions in the basal ganglia and brainstem (LS). Brain autopsy in one case revealed infarct‐like lesions in the cerebral cortex, basal ganglia and brainstem, providing further insight into the distribution of the pathological lesions in MELAS/LS overlap syndrome. This is the first report of the brain pathological changes in a patient with m.13513G>A mutation. The spatial distribution of infarct‐like lesions in the brain could explain the symptoms in MELAS/LS overlap syndrome.     

4例MELAS综合征患者的内分泌激素水平研究和文献复习

目的 探讨经基因及肌肉病理证实的4例线粒体脑肌病伴高乳酸血症和卒中样发作（mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes,MELAS）综合征患者的激素水平改变情况,并进行文献复习。 方法 2009年10月～2011年12月北京天坛医院神经内科住院的4例MELAS综合征患者,4例患者均为男性,表现为卒中样发作、癫痫、身材矮小。1例患者存在第二性征发育迟滞,1例患者合并糖尿病。2例患者进行肌肉活检,4例患者进行线粒体基因检测和内分泌激素检查。 结果 2例肌肉活检结果显示存在破碎红纤维,4例线粒体基因检测结果显示存在线粒体基因（A3243G）的突变。内分泌激素检查显示4例患者均存在内分泌异常,其中黄体生成素轻度降低1例,泌乳素增高2例,睾酮显著降低1例,甲状腺素轻度降低2例,胰岛素降低1例。 结论 MELAS综合征患者常合并内分泌异常,可存在多种内分泌激素水平的改变,包括性腺相关、甲状腺素、胰岛素等。应重视对MELAS综合征患者内分泌紊乱的诊断和治疗     

Sporadic MERRF/MELAS overlap syndrome associated with the 3243 tRNALeu(UUR) mutation of mitochondrial DNA

We studied a patient with a mitochondrial encephalomyopathy characterized by the presence of all the cardinal features of both myoclonic epilepsy and ragged-red fibers (MERRF) and mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) syndromes. Muscle biopsy showed ragged-red fibers (RRF). Some RRF were cytochrome c oxidase (COX)-negative, while some others stained positive for COX. Muscle biochemistry revealed defects of complexes I and IV of the respiratory chain. Both muscle and blood mitochondrial DNA from the patient showed the presence of the mutation at nucleotide position 3243 in the tRNA^Leu(UUR) gene and the absence of point mutations related to MERRF syndrome. The proportions of mutant mtDNA were 70% in muscle and 30% in blood. The mutation was absent in blood from all maternal relatives, in hair follicles from the mother, and in muscle from one sister of the proband. Therefore, there was no evidence of maternal inheritance. © 1996 John Wiley & Sons, Inc.     

ATP, phosphocreatine and lactate in exercising muscle in mitochondrial disease and McArdle's disease

We studied exercise-induced changes in the adenosine triphosphate (ATP), phosphocreatine (PCr), and lactate levels in the skeletal muscle of mitochondrial patients and patients with McArdle's disease. Needle muscle biopsy specimens for biochemical measurement were obtained before and immediately after maximal short-term bicycle exercise test from 12 patients suffering from autosomal dominant and recessive forms of progressive external ophthalmoplegia and multiple deletions of mitochondrial DNA (adPEO, arPEO, respectively), five patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) 3243 A-->G point mutation, and four patients with McArdle's disease. Muscle ATP and PCr levels at rest or after exercise did not differ significantly from those of the controls in any patient group. In patients with mitochondrial disease, muscle lactate tended to be lower at rest and increase more during exercise than in controls, the most remarkable rise being measured in patients with adPEO with generalized muscle symptoms and in patients with MELAS point mutation. In McArdle patients, the muscle lactate level decreased during exercise. No correlation was found between the muscle ATP and PCr levels and the respiratory chain enzyme activity.     

Aortic rupture in mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes

BACKGROUND: Microangiopathy has been well described in the brain and muscle of patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). OBJECTIVE: To describe a patient with the common A3243G/MELAS point mutation who had aortic rupture and whose mother also died of large vessel rupture. DESIGN: Case report. SETTING: Collaboration between a primary care hospital and 2 academic tertiary care hospitals. RESULTS: Histologically, there was marked disarray of the smooth muscle architecture of the aorta, and immunohistochemical staining with antibodies against the mitochondrial DNA-encoded cytochrome-C oxidase I subunit showed uniformly decreased immunostaining of the endothelial and smooth muscle cells of the aorta and vasa vasorum. Polymerase chain reaction and restriction fragment length polymorphism analysis showed that the mutation load was 40.5% in blood but 85.3% in the blood vessels. CONCLUSIONS: The severe vasculopathy in this patient is probably directly related to the high mutation load in the blood vessels. Although aortic rupture is an unusual manifestation of MELAS, it is an important potential complication in patients undergoing minor surgical procedures.     

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.     

Genotype to phenotype correlations in mitochondrial encephalomyopathies associated with the A3243G mutation of mitochondrial DNA

We studied 22 subjects carrying the A3243G point mutation of human mitochondrial DNA (mtDNA). In 14 cases the clinical phenotype was characterized by mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), while 8 patients had chronic progressive external ophthalmoplegia (CPEO). The proportion of A3243G heteroplasmy in muscle was determined by two methods: densitometry on a diagnostic restriction-fragment length polymorphism and solid-phase mini-sequencing. We found a highly significant inverse correlation between the percentage of A3243G mutation and the specific activity of complex 1, the respiratory complex with the highest number of mtDNA-encoded subunits, suggesting a direct effect of the mutation on mtDNA translation. No correlation was observed between the percentage of mutated mtDNA and the presence or absence of specific clinical features, such as stroke, ophthalmoplegia and diabetes mellitus. However, in the MELAS group the percentage of mutated mtDNA molecules was strongly correlated with the age of onset, while no such correlation was found in the CPEO group, suggesting a different time-dependent evolution of the mutation in the two groups. Finally, in contrast with other mtDNA mutations associated with ragged-red fibres (RRF), in both MELAS³²⁴³ and CPE0³²⁴³ we observed a high proportion of RRF that were positive to the histochemical reaction to cytochromec oxidase, a morphological feature that seems to be specific for the neuromuscular phenotypes associated with mutations affecting the tRNA^Leu(UUR) gene.     

Clinical and genetic features in a MELAS child with a 3271T>C mutation

A mitochondrial DNA 3271T>C point mutation was reported to be the second most common mutation (following the mutation 3243A>G) in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) in Japan. This mutation has rarely been reported in other countries. We present an 11-year-old Taiwanese girl with MELAS, who harbored the 3271T>C mutation and had manifested short stature, epilepsia partialis continua, and recurrent basal ganglia infarctions since age 6 years, and rapid intellectual regression, dysarthria, and unsteady gait since age 10 years. The proportion of 3271T>C mutant genomes in various tissues, including urinary sediments, hair follicles, blood leukocytes, and buccal mucosa cells from the patient and her mother, was analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis and quantitative real-time polymerase chain reaction. The proportion of mutant load in the patient's muscles was near 100%. Except for muscle, the highest mutation load was detected in urinary sediments of the patient by both methods. This is the first report involving mutant load analysis with quantitative real-time polymerase chain reaction in the 3271T>C mutation. The results suggest that urinary sediments may be an alternative tissue of choice which can be obtained noninvasively in the diagnosis of mitochondrial DNA 3271T>C mutations.     

"MELAS" (A3243G) mutation of mitochondrial DNA: a study of the relationships between the clinical phenotype in 19 patients and morphological and molecular data

Nineteen patients were found to harbor the mitochondrial DNA A3243G mutation associated with MELAS syndrome (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes). Eight of them had presented with stroke-like episodes and therefore had a clinical diagnosis of MELAS syndrome. The other 11 patients had no strokes and presented with generally less severe multisystemic disease. In the two groups, we compared muscle morphology, biochemical activities of muscle respiratory chain, and genetic characteristics: proportion and tissue distribution of the mutation, sequence of the 22 transfer RNA genes of the mitochondrial DNA. The proportion of mutant mtDNA in muscle was always greater than in blood. The number of patients in the two groups was too low to reach significant values. However, the patients with a MELAS syndrome presented with more severe respiratory chain abnormalities and with a proportion of the A3243G mutation that was both higher and more uniformly distributed among tissues. For symptoms others than stroke-like episodes, we did not observe any correlation with the level of mutant mtDNA in muscle. The analysis of the 22 tRNA sequences did not show differences between the two groups, and no co-inherited modifying tRNA genes could explain the variability of severity in our patients.     

Two novel mutations in PEO1 (twinkle) gene associated with chronic external ophthalmoplegia

Maintenance and replication of mitochondrial DNA require the concerted action of several factors encoded by nuclear genome. The mitochondrial helicase Twinkle is a key player of replisome machinery. Heterozygous mutations in its coding gene, PEO1, are associated with progressive external ophthalmoplegia (PEO) characterised by ptosis and ophthalmoparesis, with cytochrome c oxidase (COX)-deficient fibres, ragged-red fibres (RRF) and multiple mtDNA deletions in muscle. Here we describe clinical, histological and molecular features of two patients presenting with mitochondrial myopathy associated with PEO. PEO1 sequencing disclosed two novel mutations in exons 1 and 4 of the gene, respectively. Although mutations in PEO1 exon 1 have already been described, this is the first report of mutation occurring in exon 4.     

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.     

Neuropsychological status of mitochondrial encephalomyopathies

We studied 15 patients suffering from mitochondrial encephalomyopathies (MEM) by a neuropsychological screening procedure. Eight of the patients were diagnosed as having progressive external ophthalmoplegia (PEO), four mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), and three Kearns-Sayre syndrome (KSS). Based on test results covering memory, orientation, non-verbal intelligence, drawing, arithmetics, word list generation, trail making and digit span, only four patients were regarded as normal, two in the PEO and two in the KSS groups, while five patients were found to be demented (two patients in the PEO and three patients in the MELAS groups). Although memory problems were very common, it is concluded that no uniform pattern of neuropsychological deficits is seen in MEM, that MELAS patients apparently are severely handicapped cognitively, and that considerable mental deterioration may be seen even with normal computer-assisted tomography findings.     

Vascular pathology in chronic progressive external ophthalmoplegia with ragged-red fibers]

Vascular involvement in biopsied muscle specimens from 11 patients with chronic progressive external ophthalmoplegia (CPEO) with ragged-red fibers (RRF) was studied. Almost none of 69 intramuscular arteries examined were strongly stained with succinate dehydrogenase (SDH) except one patient who had 2 SSV (strongly SDH-reactive blood vessels) in his muscle biopsy. Although RRF and focal cytochrome c oxidase (CCO) deficiency in muscle fibers were the common histochemical changes in muscle biopsy specimens from CPEO patients, all mitochondria in both endothelial and smooth muscle cells of the arteries had normal morphology except for the two SSV and all mitochondria in the blood vessels had normal CCO activity by electron cytochemistry. The findings obtained from the present study were quite different from those in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged-red fibers (MERRF) in which the striking vascular involvement with SSV is the most common and major abnormality in muscle biopsy specimens. To study vascular involvement in mitochondrial encephalomyopathies is the one of very important clues to understand the pathophysiology of phenotypic expressions in mitochondrial encephalomyopathies.     

Intestinal pseudo-obstruction in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) associated with phenytoin therapy

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is most commonly associated with a mitochondrial DNA A to G point mutation at nucleotide 3243 (A3243G) and individuals with the disorder present a wide range of multisystemic symptoms. Seizures in MELAS patients are often intractable and require multiple antiepileptic drugs. Here we report a MELAS patient who presented with acute intestinal pseudo-obstruction following the administration of phenytoin (PHT) as an antiepileptic treatment. She presented with the first stroke-like episode at the age of 6 years and mitochondrial DNA analysis revealed A3243G with 94% mutation load in skeletal muscle. Despite treatment with phenobarbital and clobazam at the age of 16 years, she developed status epilepticus which ceased following PHT infusion. Thereafter, she was started on PHT treatment. One month later, however, she was readmitted because of remarkable abdominal distention. Although abdominal CT showed acute ileus with hepatic portal venous gas mimicking surgical emergency, the abdominal distention gradually recovered over several days following the discontinuation of PHT. Our clinical observations suggest the possibility that intestinal pseudo-obstruction in this patient related to PHT therapy. Careful clinical observation including gastrointestinal symptoms is required in the management of epilepsy in MELAS patients.     

Correlation between clinical and molecular features in two MELAS families.

We describe the clinical, morphological, biochemical presentation in two MELAS families, and correlate it with the distribution and proportion of mitochondrial DNA carrying the A to G transition at nt 3243. Family A was characterized by late onset MELAS in two members, CPEO in one, and mild CNS involvement in another. 20-61% of mtDNA of affected and unaffected individuals was mutated in muscle, 2-18% in blood. There was no obvious correlation between clinical picture and proportion of mutated mtDNA. In family B full MELAS syndrome appeared only in the third generation, but the mutation was also detected in muscle of asymptomatic individuals of the first and second generation. The proportion of mutated mtDNA in blood, and to a lesser extent in muscle, correlated with the severity of the clinical presentation. The MELAS mutation is consistently detected in all asymptomatic maternal relatives of MELAS patients. We conclude that different clinical presentations of mitochondrial encephalomyopathy may coexist in the same family, and correlation between clinical severity and molecular abnormality is not always recognizable. Presence of the MELAS mutation in muscle and blood is a necessary but not sufficient condition for the expression of the typical MELAS phenotype.     

Atypical MELAS associated with mitochondrial tRNA(Lys) gene A8296G mutation

We report on a unique patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) presenting optic atrophy, cardiomyopathy, and bilateral striatal necrosis before stoke-like episodes became apparent. Skeletal muscle total mitochondrial DNA analysis identified a heteroplasmic A to G point mutation in the tRNA(Lys) gene at position 8296. Skeletal muscle pathology revealed typical MELAS findings, including ragged-red fibers cytochrome c oxidase positive strongly succinate dehydrogenase-reactive blood vessels. Recent reports describe the 8296 mutation identified in patients with diabetes mellitus or myoclonus epilepsy with ragged-red fibers, not MELAS. We conclude that the 8296 mutation is likely to be pathogenic and that it may be not only a mutation responsible for diabetes mellitus or myoclonus epilepsy with ragged-red fibers but also for MELAS.     

Melas: an original case and clinical criteria for diagnosis.

We describe the full history and postmortem findings in one of the first identified cases of mitochondrial encephalomyopathy with stroke-like episodes (MELAS). To clarify diagnostic criteria, we analyzed 69 reported cases. The syndrome should be suspected by the following three invariant criteria: (1) stroke-like episode before age 40 yr; (2) encephalopathy characterized by seizures, dementia, or both; and (3) lactic acidosis, ragged-red fibers (RRF), or both. The diagnosis may be considered secure if there are also at least two of the following: normal early development, recurrent headache, or recurrent vomiting. There are incomplete syndromes in relatives of patients with the full syndrome and incomplete syndromes might also be encountered in sporadic cases. Some MELAS patients have features of the Kearns-Sayre syndrome (KSS) or myoclonic epilepsy with ragged-red fibers (MERRF), but none had the full KSS syndrome. In partial or confusing cases, analysis of mitochondrial DNA (mtDNA) may point to the correct diagnosis; however, not all patients with clinical MELAS have had the typical mtDNA point mutation and some patients with the mutation have clinical syndromes other than MELAS.     

Are mitochondrial DNA deletions causative in chronic progressive external ophthalmoplegia patients?

Twenty-one patients with long standing unexplained ptosis (3), chronic progressive external ophthalmoplegia (CPEO, 16) or Kearns-Sayre syndrome (KSS, 2) were studied for the presence of mitochondrial DNA (mtDNA) deletions and the major disease-associated mtDNA point mutations with the aim of correlating mitochondrial genetic abnormalities with pathogenesis in these patients. Only 52% were found to have a deletion; of these, 82% harboured the 'common deletion'. Two of 2 KSS patients and 9 of 16 CPEO patients were deletion positive. None of the 3 patients with bilateral ptosis only had a deletion. Of those patients with ragged red fibres (RRF) on histology, 69% had a deletion. No disease associated mtDNA point mutation was observed with the exception of the nucleotide (nt) 11084 A-G mutation associated with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) in a patient also harbouring the common deletion. The role of deletions in CPEO patients is discussed.     

Atypical MELAS associated with mitochondrial tRNA gene A8296G mutation

We report on a unique patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) presenting optic atrophy, cardiomyopathy, and bilateral striatal necrosis before stoke-like episodes became apparent. Skeletal muscle total mitochondrial DNA analysis identified a heteroplasmic A to G point mutation in the tRNA^Lys gene at position 8296. Skeletal muscle pathology revealed typical MELAS findings, including ragged-red fibers cytochrome c oxidase positive strongly succinate dehydrogenase-reactive blood vessels. Recent reports describe the 8296 mutation identified in patients with diabetes mellitus or myoclonus epilepsy with ragged-red fibers, not MELAS. We conclude that the 8296 mutation is likely to be pathogenic and that it may be not only a mutation responsible for diabetes mellitus or myoclonus epilepsy with ragged-red fibers but also for MELAS.