Functional consequences of the 3460-bp mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy.

Complex I is the largest of the mitochondrial respiratory chain proteins, and contains subunits encoded by both mitochondrial and nuclear genomes. Leber's hereditary optic neuropathy has been clearly linked to mutations of mitochondrial DNA complex I genes, and variable complex I functional defects have been reported. We have confirmed an approximate 60% defect in mitochondrial NADH CoQ1 reductase activity in cultured fibroblasts bearing the 3460-bp G to A mutation within the ND1 gene. However complex I-linked ATP synthesis was found to be normal in these fibroblasts. A 60% rotenone-induced decrease in complex I activity was shown to reduce ATP synthesis in normal fibroblasts, indicating that this level of complex I activity was below the threshold required to affect ATP synthesis. Although 3460 LHON mitochondria were less sensitive to rotenone inhibition, this did not explain the decreased complex I activity as the rotenone insensitive activity was not increased, nor did the inhibitor diphenyleneiodonium inhibit the NADH CoQ1 reductase activity to a greater extent. Decreased NADH cytochrome c reductase activity in cybrids homoplasmic for the 3460 LHON mtDNA mutation confirmed that the decrease in complex I activity was not specific to the assay used and was not caused by inhibitory effects of ubiquinone analogues used in the NADH CoQ1 reductase assay. These findings have important implications for our understanding of complex I dysfunction in the pathogenesis of 3460 Leber's hereditary optic neuropathy.     

肌阵挛癫痫合并破碎红纤维综合征的线粒体DNA突变特点

目的　探讨肌阵挛癫痫伴破碎红纤维综合征 ( MERRF)的分子遗传学特点。方法　用聚合酶链反应-限制片段长度多态性 ( PCR- RFL P)方法检测 6例 MERRF患者及其部分母系亲属的肌肉和 (或 )外周血细胞的mt DNA的 A8344 G点突变 ,并进行突变型 mt DNA的定量分析。结果　在 2例患者的肌肉和外周血细胞中检测到A8344 G点突变。但其母亲的外周血细胞中未能检测到此突变。这 2例 A8344 G阳性标本中 ,肌肉组织的突变型mt DNA的比例分别为 79.0 %和 86 .8%,而在外周血细胞中分别为 5 9.7%和 72 .9%,突变型 m t DNA的比例在肌肉组织中高于外周血细胞中。结论　在 MERRF患者不同组织中检测到 mt DNA A8344 G点突变 ,与国外报道一致。但国外报道 MERRF多为母系遗传 ,而我们的病例未能有此发现 ,须扩大样本量进一步分析。     

Fibrous dysplasia in a child with mitochondrial A8344G mutation

Myoclonic epilepsy associated with ragged red fibers (MERRF) syndrome is one of the major mitochondrial encephalomyopathies, with the involvement of various organs, which could be caused by mitochondrial A8344G DNA mutation. Monostotic fibrous dysplasia of bone, an asymptomatic developmental disorder, was reported to result from c-fos overexpression in osteogenic cells. Mitochondrial A8344G mutation has been shown to increase c-fos expression in a MERRF cybrid cell line. The authors describe a boy aged 10 years and 2 months with MERRF syndrome and A8344G mutation. Visual disturbance developed and deteriorated rapidly 5 months after the diagnosis of MERRF. A brain magnetic resonance imaging revealed optic nerve compression by sphenoid fibrous dysplasia, which was confirmed by histology. Fibrous dysplasia has never been mentioned in MERRF patients in the literature. This rare association may be because of underestimation, or it could be a coincidence. Care should be taken to explore the skeletal system in MERRF patients with focal symptoms.     

Distal weakness with respiratory insufficiency caused by the m.8344A > G “MERRF” mutation

The m.8344A > G mutation in the mt-tRNA^Lys gene, first described in myoclonic epilepsy and ragged red fibers (MERRF), accounts for approximately 80% of mutations in individuals with MERRF syndrome. Although originally described in families with a classical syndrome of myoclonus, ataxia, epilepsy and ragged red fibers in muscle biopsy, the m.8344A > G mutation is increasingly recognised to exhibit marked phenotypic heterogeneity. This paper describes the clinical, morphological and laboratory features of an unusual phenotype in a patient harboring the m.8344A > G ‘MERRF’ mutation. We present the case of a middle-aged woman with distal weakness since childhood who also had ptosis and facial weakness and who developed mid-life respiratory insufficiency necessitating non-invasive nocturnal ventilator support. Neurophysiological and acetylcholine receptor antibody analyses excluded myasthenia gravis whilst molecular genetic testing excluded myotonic dystrophy, prompting a diagnostic needle muscle biopsy. Mitochondrial histochemical abnormalities including subsarcolemmal mitochondrial accumulation (ragged-red fibers) and in excess of 90% COX-deficient fibers, was seen leading to sequencing of the mitochondrial genome in muscle. This identified the m.8344A > G mutation commonly associated with the MERRF phenotype. This case extends the evolving phenotypic spectrum of the m.8344A > G mutation and emphasizes that it may cause indolent distal weakness with respiratory insufficiency, with marked histochemical defects in muscle. Our findings support consideration of screening of this gene in cases of indolent myopathy resembling distal limb-girdle muscular dystrophy in which screening of the common genes prove negative.     

Genetic biochemical and pathophysiological characterization of a familial mitochondrial encephalomyopathy (MERRF).

Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome is a neuromuscular disorder characterized by mitochondrial myopathy and progressive myoclonus epilepsy. A heteroplasmic A to G transition mutation in the mitochondrial encoded tRNA(Lys) gene at nucleotide pair 8344 has been suggested to be linked to the MERRF-syndrome. We have investigated biochemically and histochemically muscle biopsies and studied the mitochondrial genomes of hair, blood and muscle tissue of a family including three cases of MERRF-syndrome as well as unaffected relatives within the maternal lineage. Sequence analysis of the mtDNAs, performed after amplification by the polymerase chain reaction (PCR), confirmed the A to G transition mutation in the tRNA(Lys) gene at position 8344. The additional point mutation at nucleotide pair 750 in the 12 S rRNA gene, which was also found by Shoffner et al. (1990), however, was absent in all investigated tissues. Quantitative analysis of the percentage of mutated mtDNA by mispairing PCR (Seibel et al., 1990) revealed variable contents in different tissues and individuals, including unaffected family members. Mitochondrial protein synthesis in cultured fibroblasts from MERRF patients revealed diminished incorporation of 35S-methionine into lysine-containing peptides.     

Myoclonus epilepsy in mitochondrial disorders

Mitochondrial disorders is a group of clinical entities associated with abnormalities of the mitochondrial respiratory chain (MRC), which carries out the oxidative phosphorylation (OXPHOS) of ADP into ATP. As the MRC is the result of genetic complementation between two separate genomes, nuclear and mitochondrial, OXPHOS failure can derive from mutations in either nuclear‐encoded, or mitochondrial‐encoded, genes. Epilepsy is a relatively common feature of mitochondrial disease, especially in early‐onset encephalopathies of infants and children. However, the two most common entities associated with epilepsy include MERRF, for Myoclonic Epilepsy with Ragged Red Fibers, and AHS, or Alpers‐Huttenlocher syndrome, also known as hepatopathic poliodystrophy. Whilst MERRF is a maternally inherited condition caused by mtDNA mutations, particularly the 8344A>G substitution in the gene encoding mt‐tRNA^Lys, AHS is typically caused by recessive mutations in POLG, encoding the catalytic subunit of polymerase gamma, the only mtDNA polymerase in humans. AHS is the most severe, early‐onset, invariably fatal syndrome within a disease spectrum, which also include other epileptogenic entities, all due to POLG mutations and including Spino‐cerebellar Ataxia and Epilepsy (SCAE). This review reports the main clinical, neuroimaging, biochemical, and molecular features of epilepsy‐related mitochondrial syndrome, particularly MERRF and AHS.     

Lipomatosis, proximal myopathy, and the mitochondrial 8344 mutation. A lipid storage myopathy?

Multiple symmetric lipomatosis (MSL) has been related in some cases to the 8344 point mutation of the tRNA-lysine gene of the mitochondrial DNA, mainly in the context of families with classic myoclonic epilepsy with ragged-red fibers (MERRF) and exceptionally in patients with proximal myopathy as the only manifestation of mitochondrial disease. We report on two families harboring the 8344 mutation. The patients presented with MSL and myopathy, expressed as limb girdle weakness in index cases and as exercise intolerance in the others. All muscle biopsies performed showed lipid storage apart from RRF and respiratory chain complexes deficiency. A possible explanation for both adipose proliferation and lipid storage myopathy in these cases is a disturbance in intermediary lipid metabolism secondary to mitochondrial respiratory chain deficiency that could be related via carnitine deficiency.     

Myoclonic epilepsy with ragged-red fibers (MERRF) syndrome: Report of a Chinese family with mitochondrial DNA point mutation in tRNALys gene

We report myoclonic epilepsy with ragged-red fibers (MERRF) syndrome in a Chinese family with confirmed mitochondrial DNA point mutation. Six members of the family including the grandmother, two siblings, and three grandchildren were affected. Among them, action myoclonus was seen in five; short stature, muscle weakness, and mental retardation in four; lactic acidosis, hearing impairment, and ataxia in two; and seizures in one. Muscle biopsy from two affected siblings revealed ragged-red fibers and abundant subsarcolemmal mitochondria with paracrystalline inclusions. Pedigree analysis suggests a maternal transmission. Analysis of mitochondrial DNA showed a point mutation from A to G at the 8344th nucleotide position located in the tRNA_Lys gene. To our knowledge, this is the first report of MERRF syndrome with such genetic defect from a Chinese family. The present and previous reports support the notion that mitochondrial DNA point mutation at the 8344th nucleotide position is the most common cause of MERRF syndrome. © 1994 John Wiley & Sons, Inc.     

Autopsied case with MERRF/MELAS overlap syndrome accompanied by stroke‐like episodes localized to the precentral gyrus

We present an autopsied case with A8344G‐mutated myoclonus epilepsy with ragged red fibers (MERRF)/mitochondrial encephalomyopathy with lactic acidosis and stroke‐like episodes (MELAS) overlap syndrome accompanied by stroke‐like episodes localized to the precentral gyrus. A 16‐year‐old Japanese woman suddenly experienced repetitive consciousness disturbances with increased serum lactate and creatine kinase levels. Magnetic resonance imaging showed abnormal intensity of bilateral precentral gyrus. She was clinically diagnosed as having a mitochondrial disorder and the A8344G mutation was detected in mitochondrial DNA. At 17 years of age, she died from congestive heart failure secondary to a third episode of lactic acidosis. Neuropatho‐logically, multifocal laminar necrosis, which is responsible for stroke‐like episodes in MELAS, was seen in the frontal cortex including the precentral gyrus, but there was no neuronal loss and gliosis in the basal ganglia, cerebellum, and brainstem, which were compatible with MERRF. Hypertrophy of the vascular smooth muscle and choroidal epithelium were seen, and were strongly visualized by an anti‐mitochondrial antibody. Skeletal muscles showed uneven muscular diameters, increased central nuclei, and ragged red fibers (RRFs). Decreased cytochrome c oxidase (COX) activity and strongly succinate dehydrogenase (SDH)‐reactive blood vessels were also noted. Stroke‐like episodes in MERRF/MELAS overlap syndrome are thought to be rare in the frontal cortex including the precentral gyrus. Only two cases of MERRF/MELAS overlap syndrome with A8344G mutation, including this case, have shown stroke‐like episodes in the frontal lobes. Other than the A8344G mutation and frontal lobe involvement, they had a high degree of similarity in terms of presence of RRFs, gastrointestinal dysfunction, and lack of typical MERRF neuropathology. In conclusion, this is an important case describing the clinical spectrum associated with A8344G‐mutated MERRF/MELAS overlap syndrome.     

MERRF: a model disease for understanding the principles of mitochondrial genetics

The principles of mitochondrial genetics have evolved over the past 20 years. Careful identification of large pedigrees that were consistent with maternal inheritance has permitted detailed clinical and genetic investigations. Myoclonic epilepsy and ragged-red fiber (MERRF) disease has been a model disease for the application of these principles. MERRF is caused by an A to G mutation of the mitochondrial tRNA(Lys) at position 8344. The mutation is maternally inherited and heteroplasmic. Disease manifestations are dependent on replicative segregation of mutant and wild type mitochondrial DNAs and on the threshold effect. Characterization of the clinical, physiological, biochemical, and genetic manifestations of this disease has provided a better understanding of how to diagnose and manage oxidative phosphorylation diseases which are caused by mutations in the mitochondrial DNA.     

Late-onset Leigh syndrome with myoclonic epilepsy with ragged-red fibers

We report the case of a boy with myoclonic epilepsy with ragged-red fibers (MERRF) who had astatic seizures since 2 years of age and later developed ataxia, absence seizures, and myoclonus. Almost homoplasmic A8344G mutation of mitochondrial DNA (m.8344A>G mutation) was detected in lymphocytes. He developed late-onset Leigh syndrome (LS) when he contracted pneumonia at 6 years. He developed bulbar palsy and deep coma. MRI demonstrated lesions in the brainstem, basal ganglia, and cerebral cortex. Three similar cases have been reported; two carried the almost-homoplasmic m.8344A>G mutation in muscle tissue. These suggested that almost homoplastic m.8344A>G mutation developed clinical phenotype of MERRF in the early stage and late-onset Leigh syndrome in the late course of the disease.     

Adrenal insufficiency in a child with MELAS syndrome

Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) are established subgroups of mitochondrial encephalomyopathy. m.3243A > G a common point mutation is detected in tRNA in majority of patients with MELAS phenotype whereas m.8344A > G point mutation in tRNA is observed, in MERRF phenotype. Adrenal insufficiency has not been reported in mitochondrial disease, except in Kearns-Sayre Syndrome (KSS), which is a mitochondrial deletion syndrome. We report an unusual presentation in a five year old boy who presented with clinical phenotype of MELAS and was found to have m.8344A > G mutation in tRNA. Addison disease was identified due to hyperpigmentation of lips and gums present from early childhood. This is the first report describing adrenal insufficiency in a child with MELAS phenotype.     

Mitochondrial DNA from platelets of sporadic ALS patients restores normal respiratory functions in rho(0) cells

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, which affects the anterior horn cells of the spinal cord and cortical motor neurons. A pathophysiological role for mtDNA mutations was postulated based on the finding that cybrids obtained from mitochondria of sporadic ALS patients exhibited impaired respiratory chain activities, increased free radical scavenging enzymes, and altered calcium homeostasis. To date, however, no distinct mtDNA alterations associated with ALS have been reported. Therefore, we reexamined the hypotheses that mtDNA mutations accumulate in ALS and that cybrids generated from ALS patients' blood have impaired mitochondrial respiration. Cybrid cell lines were generated from 143B osteosarcoma rho(0) cells and platelet mitochondria of sporadic ALS patients or age-matched controls. We found no statistically significant differences in mitochondrial respiration between ALS and control cybrids, even when the electron transport chain was stressed with low concentrations of respiratory chain inhibitors. Mitochondrial respiratory chain enzyme activities were also normal in ALS cybrids, and there was no increase in free radical production. Therefore, we showed that mtDNA from platelets of ALS patients was able to restore normal respiratory function in rho(0) cells, suggesting that the presence of mtDNA mutations capable of affecting mitochondrial respiration was unlikely.     

MERRF family with 8344 mutation in tRNA (lys). Evidence of a mitochondrial vasculopathy in muscle biopsies

This article reports a new MERRF family. The mother, regarded as suffering from Ramsay-Hunt Syndrome, and her three daughters, had the same clinical pattern: myoclonic epilepsy and ataxia. Two daughters were studied on morphological, biochemical and molecular genetic levels. Muscle biopsies showed ragged-red fibres and mitochondrial vasculopathy. Arterioles were strongly SDH-reactive and COX-negative. By electron microscopy, abnormal mitochondria were observed in skeletal muscle fibres, in smooth muscle fibres of intramuscular vessels and in sweat gland epithelium. The study of the respiratory chain showed complex IV and I + IV deficiency, respectively. Mitochondrial tRNA (lys) mutation at position 8344 was pointed out as previously reported in the MERRF syndrome.     

Myoclonus in mitochondrial disorders

Myoclonus is a possible manifestation of mitochondrial disorders, and its presence is considered, in association with epilepsy and the ragged red fibers, pivotal for the syndromic diagnosis of MERRF (myoclonic epilepsy with ragged red fibers). However, its prevalence in mitochondrial diseases is not known. The aims of this study are the evaluation of the prevalence of myoclonus in a big cohort of mitochondrial patients and the clinical characterization of these subjects. Based on the database of the “Nation‐wide Italian Collaborative Network of Mitochondrial Diseases,” we reviewed the clinical and molecular data of mitochondrial patients with myoclonus among their clinical features. Myoclonus is a rather uncommon clinical feature of mitochondrial diseases (3.6% of 1,086 patients registered in our database). It is not strictly linked to a specific genotype or phenotype, and only 1 of 3 patients with MERRF harbors the 8344A>G mutation (frequently labeled as “the MERRF mutation”). Finally, myoclonus is not inextricably linked to epilepsy in MERRF patients, but more to cerebellar ataxia. In a myoclonic patient, evidences of mitochondrial dysfunction must be investigated, even though myoclonus is not a common sign of mitochondriopathy. Clinical, histological, and biochemical data may predict the finding of a mitochondrial or nuclear DNA mutation. Finally, this study reinforces the notion that myoclonus is not inextricably linked to epilepsy in MERRF patients, and therefore the term “myoclonic epilepsy” seems inadequate and potentially misleading. © 2014 International Parkinson and Movement Disorder Society     

以光敏性癫痫为主要表现的肌阵挛性癫痫伴肌肉破碎红纤维综合征一家系分析

目的研究1个以光敏性癫痫为主要表现的肌阵挛性癫痫伴肌肉破碎红纤维综合征(MERRF)家系的临床特点、遗传学特征。方法整理一个以光敏性癫痫为主要表现的肌阵挛性癫痫伴肌肉破碎红纤维综合征家系的临床表现、辅助检查及影像学资料,分析其临床特点和遗传特征。结果该家系呈母系遗传,共4人(包括先证者3个同辈,1个子代)出现肌阵挛表现,先证者以光敏性癫痫为主要表现,其肌肉活检可见典型的破碎红纤维(RRF),先证者的线粒体DNA提示8344位点由A突变为G。结论 MERRF家系少见,可以光敏性肌阵挛癫痫为主要表现。     

Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy

A G to A transition at nucleotide 11778 in the ND4 subunit gene of complex I was the first point mutation in the mitochondrial genome linked to a human disease. It causes Leber Hereditary Optic Neuropathy, a disorder with oxidative phosphorylation deficiency. To overcome this defect, we made a synthetic ND4 subunit compatible with the "universal" genetic code and imported it into mitochondria by adding a mitochondrial targeting sequence. For detection we added a FLAG tag. This gene was inserted in an adeno-associated viral vector. The ND4FLAG protein was imported into the mitochondria of cybrids harboring the G11778A mutation, where it increased their survival rate threefold, under restrictive conditions that forced the cells to rely predominantly on oxidative phosphorylation to produce ATP. Since assays of complex I activity were normal in G11778A cybrids we focused on changes in ATP synthesis using complex I substrates. The G11778A cybrids showed a 60% reduction in the rate of ATP synthesis. Relative to mock-transfected G11778A cybrids, complemented G11778A cybrids showed a threefold increase in ATP synthesis, to a level indistinguishable from that in cybrids containing normal mitochondrial DNA. Restoration of respiration by allotopic expression opens the door for gene therapy of Leber Hereditary Optic Neuropathy.     

Bilateral putaminal necrosis associated with the mitochondrial DNA A8344G myoclonus epilepsy with ragged red fibers (MERRF) mutation: an infantile case

Myoclonus epilepsy with ragged red fibers (MERRF) is one of the major mitochondrial encephalomyopathies. Its main clinical features are myoclonus epilepsy, ataxia, and myopathy with ragged red fibers. Whereas there is a close correlation between MERRF syndrome and the A8344G mutation of mitochondrial DNA, the reverse is not true. In fact, this mutation is also responsible for various other syndromes, such as Leigh syndrome, spinocerebellar degeneration, atypical Charcot-Marie-Tooth disease, and multiple truncal lipomas. We describe a child with the A8344G mutation of mitochondrial DNA and an unusual clinical, neuroradiologic, and biochemical phenotype, characterized by early-onset, nonprogressive cerebellar ataxia, and subclinical myoclonias in association with bilateral putaminal necrosis on magnetic resonance imaging and a reduction in complex V activity. Our case confirms the existence of a relationship between alteration in adenosine triphosphatase activity and basal ganglia involvement. We recommend that the possibility of a mitochondrial pathology should always be taken into consideration in the presence of bilateral symmetric lesions of the basal ganglia, even when the typical clinical picture is lacking. (J Child Neurol 2006;21:79-82).     

The A8344G mutation in mitochondrial DNA associated with stroke-like episodes and gastrointestinal dysfunction

We report an unusual case of encephalo-entero-myopathy associated with the A8344G mutation in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). This patient had mitochondrial myopathy, multiple lipomatosis, mild hearing loss, stroke-like episodes, and paralytic ileus, but she lacked the canonical clinical features of MERRF, myoclonus, epilepsy, or ataxia. We conducted genetic, biochemical, histochemical, and immunohistochemical studies in skeletal muscle, brain, intestine, and lipoma tissue. The mutation was abundant in all tissues, and cytochrome c oxidase (COX) activity was selectively decreased in brain and small intestine. COX deficiency was also documented histochemically and immunohistochemically in the small intestine, suggesting that mitochondrial dysfunction played a role in the pathogenesis of paralytic ileus. This case illustrates an unusual and dramatic clinical phenotype of the A8344G mutation, characterized by stroke-like episodes and acute ileus.