Progressive fat replacement of muscle contributes to the disease mechanism of patients with single,large-scale deletions of mitochondrial DNA

Muscle dysfunction in mitochondrial myopathy is predominantly caused by insufficient generation of energy. We hypothesise that structural changes in muscles could also contribute to their pathophysiology. The aims of this study were to determine fat fractions and strength in selected muscles in patients with chronic progressive external ophthalmoplegia (CPEO), and compare progression of muscle fat fraction with age in individuals with CPEO vs. healthy controls and patients with the m.3243A>G mutation of mitochondrial DNA (mtDNA). Seventeen patients with CPEO and single large-scale deletions of mtDNA, 52 healthy controls, and 12 patients carrying the m.3243A>G mtDNA mutation were included. Muscle fat fractions were measured from cross-sections of paraspinal and leg muscles. Peak muscle strength was assessed from a static dynamometer. There was a direct correlation between age and fat fraction in all muscle groups in CPEO patients and healthy controls (p < 0.05). Analysis of covariance showed a higher progression rate of fat replacement in CPEO patients vs. healthy controls in studied muscle groups (p < 0.05). Patients with the m.3243A>G mutation had slower progression rates of fat replacement. Muscle strength decreased with increasing muscular fat fraction in CPEO patients, no correlation was seen in other groups. This indicates that structural muscle changes contribute to the phenotype of older patients affected by CPEO and large-scale deletions. It should therefore be considered, along with known energy deficiencies, as the cause of exercise intolerance.     

Limited diagnostic value of enzyme analysis in patients with mitochondrial tRNA mutations

We evaluated the diagnostic value of respiratory chain (RC) enzyme analysis of muscle in adult patients with mitochondrial myopathy (MM). RC enzyme activity was measured in muscle biopsies from 39 patients who carry either the 3243A>G mutation, other tRNA point mutations, or single, large‐scale deletions of mtDNA. Findings were compared with those obtained from asymptomatic relatives with the 3243A>G mutation, myotonic dystrophy patients, and healthy subjects. Plasma lactate concentration, maximal oxygen uptake, and ragged‐red fibers/cytochrome c–negative fibers in muscle were also determined. Only 10% of patients with the 3243A>G point mutation had decreased enzyme activity of one or more RC complexes, whereas this was the case for 83% of patients with other point mutations and 62% of patients with deletions. Abnormal muscle histochemistry was found in 65%, 100%, and 85% of patients, respectively, in these three groups. The results indicate that RC enzyme analysis in muscle is not a sensitive test for MM in adults. In these patients, abnormal muscle histochemistry appears to be a better predictor ofMM. Muscle Nerve, 2010     

Neuroimaging characteristics in mitochondrial encephalopathies associated with the m.3243A>G MTTL1 mutation

Stroke-like lesions (SLL) are common radiological findings in patients with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (SLE; MELAS) harboring the m.3243A>G MTTL1 mutation. Imaging patterns in the m.3243A>G mutation carriers with encephalopathies lacking SLE have not been systematically examined to date. The aim of this study was to analyze brain imaging findings in encephalopathies associated with the m.3243A>G mutation irrespective of the presence or absence of SLE. Brain MRI and cranial CT scans from 11 m.3243A>G mutation carriers with encephalopathies were analyzed by two neuroradiologists in consensus. We evaluated stroke-like lesions (SLL), deep grey matter (DGM) changes on T1- and T2-weighted MR images, calcification on CT, brain atrophy, and white matter (WM) changes. SLL were present in all patients showing the full MELAS phenotype with SLE (4/11). Seven patients did not show SLE. DGM changes with T1 hyperintensity and T2 hypointensity were a distinctive finding in most patients (7/11) and present in the majority of m.3243A>G mutation carriers lacking SLE (5/7). DGM changes were also seen in half of our MELAS patients with SLL (2/4), though less pronounced. Brain atrophy was a prominent finding in general and accentuated in the cerebellum. In contrast, WM changes were rather mild and more prevalent and pronounced in MELAS. Our data stress that the distinction between MELAS with SLE and m.3243A>G mutation carriers lacking SLE is rather artificial. In clinical practice, mitochondrial disorders associated with the m.3243A>G mutation should be taken into consideration in encephalopathies with DGM changes, even when SLE and SLL are lacking.     

Acute mitochondrial myopathy with respiratory insufficiency and motor axonal polyneuropathy

Background: Mitochondrial myopathies (MMs) are mainly presented with chronic muscle weakness and accompanied with other syndromes. MM with acute respiratory insufficiency is rare. Aims: To reveal the clinical, pathological and molecular characteristics of a life-threatening MM. Methods: Muscle biopsy and enzyme staining were performed in skeletal muscles. Mitochondrial DNA (mtDNA) sequencing was analyzed and heteroplasmy were quantified by pyrosequencing. Results: All three patients had tachycardia, acute lactic acidosis, dyspnea and sudden severe muscle weakness. Two patients had calf edema and abdominal pain, and one had a heart attack. Electromyography in two patients showed dramatically decreased axonal amplitudes of motor nerves. Muscle biopsies showed ragged red fibers and dramatic mitochondrial abnormality. A mtDNA m.3243A>G mutation was identified in Patient 1 (mutation load: 29% in blood and 73% in muscle) and Patient 3 (79% in blood and 89% in muscle). A mtDNA m.8344A>G mutation was found in Patient 2 (mutation load 80.4% in blood). Conclusion: MM characterized by lactic acidosis, respiratory failure and acute motor axonal neuropathy is life threatening.     

Phenotypes and mitochondrial DNA substitutions in families with A3243G mutation

OBJECTIVE: To clarify the relationship between mitochondrial DNA (mtDNA) sequence variations and phenotypes in patients with A3243G mutation. MATERIALS AND METHODS: We studied whole mtDNA sequences in two families with A3243G mutation and characteristic clinical features. Two brothers in Family 1 had shown thiamine deficiency and mitochondrial myopathy without central nervous system involvement. In Family 2, a 16-year-old woman showed the symptoms of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Her mother had had diabetes mellitus and died at the age of 42. The proportion of A3243G mtDNA in blood was 87 and 89% in the patients of Family 1, and 25% in the patient and less than 5% in the mother of Family 2. RESULTS: The mtDNA analysis revealed the following homoplasmic substitutions: T1520C and C12153T found only in Family 1, and A15954G found only in Family 2. These substitutions were not detected in seven other MELAS patients or in 50 controls. CONCLUSION: These substitutions might be specific to these families and could be one of the factors that modulate their clinical features together with the A3243G mutation.     

MELAS phenotype associated with m.3302A>G mutation in mitochondrial tRNA gene

The m.3302A>G mutation in the mitochondrial tRNA^Leu(UUR) gene has been identified in only 12 patients from 6 families, all manifesting adult-onset slowly progressive myopathy with minor central nervous system involvement. An 11-year-old boy presented with progressive proximal-dominant muscle weakness from age 7 years. At age 10, he developed recurrent stroke-like episodes. Mitochondrial myopathy, encephalopathy, lactic acidosis, plus stroke-like episodes (MELAS) was diagnosed by clinical symptoms and muscle biopsy findings. Mitochondrial gene analysis revealed a heteroplasmic m.3302A>G mutation. Histological examination showed strongly SDH reactive blood vessels (SSVs), not present in previous cases with myopathies due to the m.3302A>G mutation. These findings broaden the phenotypic spectrum of this mutation.     

The m.3243A>G mitochondrial DNA mutation and related phenotypes. A matter of gender?

The m.3243A>G “MELAS” (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) mutation is one of the most common point mutations of the mitochondrial DNA, but its phenotypic variability is incompletely understood. The aim of this study was to revise the phenotypic spectrum associated with the mitochondrial m.3243A>G mutation in 126 Italian carriers of the mutation, by a retrospective, database-based study (“Nation-wide Italian Collaborative Network of Mitochondrial Diseases”). Our results confirmed the high clinical heterogeneity of the m.3243A>G mutation. Hearing loss and diabetes were the most frequent clinical features, followed by stroke-like episodes. “MIDD” (maternally-inherited diabetes and deafness) and “PEO” (progressive external ophthalmoplegia) are nosographic terms without any real prognostic value, because these patients may be even more prone to the development of multisystem complications such as stroke-like episodes and heart involvement. The “MELAS” acronym is convincing and useful to denote patients with histological, biochemical and/or molecular evidence of mitochondrial disease who experience stroke-like episodes. Of note, we observed for the first time that male gender could represent a risk factor for the development of stroke-like episodes in Italian m.3243A>G carriers. Gender effect is not a new concept in mitochondrial medicine, but it has never been observed in MELAS. A better elucidation of the complex network linking mitochondrial dysfunction, apoptosis, estrogen effects and stroke-like episodes may hold therapeutic promises.     

MELAS- and kearns-sayre-type with myopathy and autoimmune polyendocrinopahy

A 35-year-old woman with features of Kearns-Sayre syndrome consisting of progressive ptosis, ophthalmoparesis, mitochondrial myopathy, and pigmentary retinopathy also had autoimmune polyglandular syndrome type I1 (Addison's disease, autoimmune insulin-dependent diabetes meuitus, Hashimoto's thyroiditis, and primary ovarian failure). There was no history of similarly affected relatives. Analysis of muscle mitochondrial DNA (mtDNA) revealed a 2,532-bp deletion of the type seen in Kearns-Sayre syndrome as well as a heteroplasmic A3243G mutation in the tRNA-Leu(UUR) gene of the type seen in mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). The patient's blood and her mother's blood harbored the A3243G mutation but not the deletion, and the maternal grandmother's blood had neither mutation. In muscle, the species of mtDNA harboring the deletion was exclusively associated with the species harboring the A3243G mutation, suggesting that the point mutation predisposed to the large-scale deletion. The mtDNA species with both mutations accounted for 88% of total muscle mtDNA. Other and as yet unrecognized point mutations in mtDNA might also be associated with, and possible causally related to, largescale mtDNA deletions.     

Mitochondrial myopathy and familial thiamine deficiency

We studied two siblings with a mitochondrial myopathy, familial thiamine deficiency, and an A3243G mutation of the mitochondrial DNA (mtDNA). The elder brother (patient 1, now 36 years old) developed myopathy and beriberi heart at 20 years of age. Thiamine therapy resolved the cardiac symptoms and hyperpyruvicemia and improved the myopathy. The younger brother presented aged 19 years with a myopathy (patient 2, now 35 years old). Thiamine deficiency was present in the siblings and parents, and ragged-red fibers (RRFs) were noted in muscle biopsies from the siblings. Analysis 17 years later demonstrated thiamine malabsorption and an A3243G mutation of the mtDNA in both siblings and their mother, progressive myopathy, and an increased number of RRFs and elevated serum CKMB activity in patient 1. Thiamine treatment decreased the serum concentrations of lactate and pyruvate in patient 2, but not patient 1. The role of thiamine in mitochondrial dysfunction caused by an electron transfer disorder in the setting of A3243G mtDNA mutation is discussed.     

肢带型线粒体肌病一个家系的临床和病理特点研究

目的研究肢带型线粒体肌病一个家系中患者的临床和骨骼肌病理特点。方法对一组以进行性肌肉无力为主要特点的家系中的3名患者进行临床资料收集及肌肉活检病理分析,对家系成员行线粒体基因检测。结果该家系患者发病年龄为30~47岁,主要表现为躯干肌及四肢近端肌无力和运动耐力下降,晚期出现呼吸肌受累。血肌酸激酶水平轻度升高,血乳酸升高,电生理检查结果示肌源性损害。骨骼肌病理检查显示3名患者均存在典型的破碎红纤维(RRF),且患者病程越长(分别为2年、4年和14年),肌无力严重程度越重,RRF数量越多(分别为5%、10%和30%)。细胞色素c氧化酶(COX)染色中RRF以深染为主,也可见阴性的RRF。琥珀酸脱氢酶(SDH)染色均未发现琥珀酸脱氢酶高反应性血管(SSVs)。基因检测发现该家系存在mt DNA A3243G突变。结论肢带型线粒体肌病患者选择性累及躯干和四肢近端肌群,骨骼肌中的RRF数量与患者病情进展程度呈正相关。     

The mitochondrial DNA A3243G mutation in Portugal: clinical and molecular studies in 5 families.

Out of 90 Portuguese patients with mitochondrial cytopathy, six harbored the A3243G mutation in the mtDNA tRNA(Leu(UUR)) gene ('MELAS mutation'). They had heterogeneous clinical features, including myopathy with stroke-like episodes, progressive external ophthalmoparesis, diabetes mellitus, and subacute encephalopathy. Histochemical and biochemical analyses of muscle biopsies showed abundant ragged-red fibers reacting positively with the cytochrome oxidase stain, and decreased respiratory chain enzyme activities. On average, the proportion of mutated mtDNA was 67% (20-88%) in tissues from patients and 21% (0-49%) in blood from 20 maternal relatives. The proportion of mutated mitochondrial genomes in muscle did not correlate with clinical presentation or duration of disease. This study, the first in Portuguese patients, confirms the frequent occurrence of the A3243G mutation in patients with mitochondrial diseases, and emphasises the usefulness of genetic testing in reaching a correct diagnosis.     

The pathogenic m.3243A>T mitochondrial DNA mutation is associated with a variable neurological phenotype

The m.3243A>G point mutation in the mitochondrial tRNA^Leu(UUR) (MTTL1) gene is a common cause of mitochondrial DNA disease and is associated with a variety of clinical presentations. A different mutation occurring at the same site – an m.3243A>T transversion – is less prevalent, but has previously been observed in two patients with encephalopathy and lactic acidosis. We report the investigations of a further two patients with the m.3243A>T mutation who presented with either a chronic progressive external ophthalmoplegia (CPEO) phenotype or sensorineural hearing loss, with single fibre mutation studies confirming segregation of the m.3243A>T mutation with COX deficiency.     

Epidemiology and characteristics of occipital brain infarcts in young adults in southwestern Finland

Occipital stroke and occipital epilepsy are possible manifestations of mitochondrial diseases. A previous study in northern Finland suggested a frequency of 10% for mitochondrial disorder in young patients with stroke. Here we studied the epidemiology of occipital brain infarcts in a defined population in southwestern Finland. Patients diagnosed with brain infarct or visual field defect with onset at the ages of 18–45 years were identified from the discharge files at the Turku University Hospital. We further ascertained those patients with an occipital brain infarct in brain imaging or homonymous hemianopia with no signs of other etiology in brain imaging. We reviewed the clinical data for known stroke risk factors and analyzed samples for the m.3243A > G and m.8344A > G mutations in mitochondrial DNA (mtDNA), and determined mtDNA haplogroups and five common mutations in the gene encoding polymerase γ (POLG1). Migraine was more common in young patients with occipital brain infarct than in the general population, especially among women. None of the patients harboured the m.3243A > G or m.8344A > G mutation in mtDNA or any of the five common mutations in POLG1. Interestingly, 17% of the men and 33% of the women belonged to the mtDNA haplogroup Uk, while its frequency in the general population is 17%. Our results suggest that mtDNA haplogroup Uk is associated with increased risk of occipital stroke in young women. POLG1 mutations have been associated with occipital epilepsy, but we did not find the common mutations in patients with occipital stroke.     

Cardiomyopathy is common in patients with the mitochondrial DNA m.3243A>G mutation and correlates with mutation load

Although neuromuscular clinical features often dominate the clinical presentation of mitochondrial disease due to the m.3243A>G mitochondrial DNA (mtDNA) mutation, many patients develop cardiac failure, which is often overlooked until it reaches an advanced stage. We set out to determine whether cardiac complications are sufficiently common to warrant prospective screening in all mutation carriers. Routine clinical echocardiography and 3 Tesla cardiac MRI were performed on ten m.3243A>G mutation carriers and compared to age and gender matched controls, with contemporaneous quadriceps muscle biopsies to measure respiratory chain activity and mtDNA mutation levels. Despite normal echocardiography, all ten m.3243A>G mutation carriers had evidence of abnormal cardiac function on MRI. The degree of cardiac dysfunction correlated with the percentage level of mutant mtDNA in skeletal muscle. Sub-clinical cardiac dysfunction was a universal finding in this study, adding weight to the importance of screening for cardiac complications in patients with m.3243A>G. The early detection of cardiac dysfunction with MRI opens up opportunities to prevent heart failure in these patients through early intervention.     

Association of the MELAS m.3243A>G mutation with myositis and the superiority of urine over muscle,blood and hair for mutation detection

A patient with a known family history of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) due to the MT-TL1 m.3243A>G mutation presented with mild myalgia and very minor upper limb proximal muscle weakness. Muscle histology revealed low levels of cytochrome oxidase-negative fibres and non-specific myositis. Using the last “hot cycle” polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), the MELAS MT-TL1 m.3243A>G mutation was only detected in urine, and not in hair, blood or skeletal muscle. This report highlights the need to screen various tissues to achieve an accurate mitochondrial genetic diagnosis and suggests the likelihood of myositis arising secondary to the MELAS MT-TL1 m.3243A>G mutation.     

Tissue mosaicism in the skeletal muscle and sural nerve biopsies in the MELAS syndrome

We describe a clinically full-blown MELAS patient, who had an A3243G point mutation of mitochondrial DNA (mtDNA) in muscle and blood cells, and his family members. From the proband two muscle biopsies from the vastus lateralis muscle were analysed; one had typical ragged red fibers and focal cytochrome c oxidase deficiency and the other was completely normal. He also had a peripheral neuropathy confirmed by nerve conduction velocity and sural nerve biopsy studies. Axonal degeneration, relative loss of large myelinated fibers and paracrystalline inclusion bodies in the Schwann cells were noted. Intriguingly, the A3243G mutation of mtDNA was not found in the sural nerve biopsy. Therefore, we conclude that tissue mosaicism is present in the muscle fibers and that the mtDNA mutation may not be detected in the nerve involved as proved by pathology. We also suggest that the involvement of specific tissues in patients with mitochondrial diseases should be further determined by single fiber mtDNA analysis.     

"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.     

Muscle structural changes in mitochondrial myopathy relate to genotype

Abstract. It is well known that morphological changes at the cellular level occur in muscle of patients with mitochondrial myopathy (MM), but changes in muscle structure with fat infiltration and gross variation of muscle fiber size with giant fibers, normally encountered in the muscular dystrophies, have typically not been associated with mitochondrial disease. We investigated gross and microscopic muscle morphology in thigh muscles by muscle biopsy and MRI in 16 patients with MM, and compared findings with those obtained in muscular dystrophy patients and healthy subjects. Changes of muscle architecture, similar to those found in the group of muscular dystrophy patients occurred consistently in patients with a high mutation load for single, largescale deletions of mtDNA, but were absent in all patients with the 3243AG mtDNA point mutation. Dystrophic changes of muscle architecture were also present in one MM patient with a unique, sporadic mutation in the mtDNA tRNA^Met gene. These findings provide evidence that morphological changes in muscle of MM patients are common and may resemble those of muscular dystrophies, but that development of dystrophic-like changes in muscle relate to genotype.     

MERRF/MELAS overlap syndrome in a family with A3243G mtDNA mutation

Four members of a family were found to carry the A3243G mtDNA mutation. Clinical features varied from typical MELAS to myoclonic epilepsy to simple deafness without neurological signs. Several other members of the family had symptoms consistent with a mitochondrial disease. Muscle biopsy in 3 of the 4 patients showed the most prominent mitochondrial alterations with partial deficiency of cytochrome c oxidase in the case with the mildest phenotype. Mitochondrial DNA analysis detected a variable percentage of A3243G mutation, roughly correlating with the phenotype. The interesting feature of the family lies in the great intrafamilial variability of the severity of clinical expression, encompassing MELAS and MERRF features, associated with the A3243G mtDNA mutation. A search for the most common mtDNA mutations is recommended in all patients featuring incomplete MELAS or MERRF syndromes and in all familial cases presenting minimal clinical signs.     

Involvement of nervous system in maternally inherited diabetes and deafness (MIDD) with the A3243G mutation of mitochondrial DNA

OBJECTIVES: The A3243G mutation of mitochondrial DNA (mtDNA) has been associated with maternally inherited diabetes and deafness (MIDD) in a number of reports; however, the involvement of the nervous system has rarely been mentioned, prompting this exploration of the manifestation of neurological disorders in MIDD cases. MATERIAL AND METHODS: We investigated four generations of a large Taiwanese family in which MIDD is manifest. We conducted a series of clinical examinations, including computed tomography (CT) and magnetic resonance imaging (MRI) of the head, brain 99mTc-HMPAO single photon emission computed tomography (SPECT), cognitive function tests, and nerve conduction velocity (NCV) studies. Blood levels of creatine kinase (CK) and lactate, pathology of muscle biopsy samples and proportions of mutant mtDNA in blood cells, hair follicles, muscle and skin were also analyzed. Mean follow-up period was 4 years. RESULTS: The patients exhibited the clinical features of diabetes mellitus including sensorineural hearing loss, short stature, and/or histories of spontaneous abortion. No stroke-like episodes were reported. Analysis for mtDNA revealed that the A3243G mutation existed in 11 members (6 symptomatic and 5 asymptomatic members) of this MIDD-prone family, with the proportion of mutant mtDNA ranging from 21% to 47% in leukocytes. Head CT revealed diffuse brain atrophy for all 6 (100%) patients examined and bilateral basal ganglia calcification in 4 of 6 (67%) patients. Brain 99mTc-HMPAO SPECT revealed diminished uptake in the bilateral parieto-occipital or occipital regions for all 6 tested patients, cognitive function for these patients was normal. Results of head CT and SPECT were normal in one asymptomatic member of the family. One muscle biopsy revealed abundant ragged-red fibers with modified Gomori-trichrome stain. Muscle-enzyme activity and serum-lactate levels were normal. CONCLUSION: We have demonstrated that a wide spectrum of sub clinical pathologies of the central nervous system and muscle are present for this MIDD-prone family, none of whom developed typical MELAS during the 4-year period of follow-up study.