A tandem duplication in the D-loop of human mitochondrial DNA is associated with deletions in mitochondrial myopathies.

About 40 per cent of patients with mitochondrial myopathies have two populations of mitochondrial DNA (mtDNA) in muscle, one of which is deleted. All patients with single mtDNA deletions and neurological disease are sporadic cases, suggesting that deletions arise as fresh mutational events. We have detected a low abundance heteroplasmic tandem duplication involving the displacement loop of mtDNA in 18 of 58 patients with deletions and 5/5 of their mothers, but not in normal subjects. The location of the duplication to a region that controls both replication and transcription of mtDNA could explain features suggesting mild mitochondrial dysfunction in the muscle biopsies of three patients' mothers, and a predisposition to deletion.

Mitochondrial encephalomyopathies: the enigma of genotype versus phenotype.

Over the past decade a large body of evidence has accumulated implicating defects of human mitochondrial DNA in the pathogenesis of a group of disorders known collectively as the mitochondrial encephalomyopathies. Although impaired oxidative phosphorylation is likely to represent the final common pathway leading to cellular dysfunction in these diseases, fundamental issues still remain elusive. Perhaps the most challenging of these is to understand the mechanisms which underlie the complex relationship between genotype and phenotype. Here we examine this relationship and discuss some of the factors which are likely to be involved.

Mitochondrial DNA (mtDNA) diseases: correlation of genotype to phenotype.

This study examines the relationship of genotype to phenotype in 14 unselected patients who were found to harbour the A3243G transition in the mitochondrial transfer RNALeu(UUR) gene commonly associated with the syndrome of mitochondrial encephalopathy, lactic acidosis and strokes (MELAS). Only 6 of the 14 cases (43%) had seizures and recurrent strokes, the core clinical features of the MELAS phenotype. Of the remaining cases, four had an encephalomyopathy with deafness, ataxia and dementia, two had syndromes with progressive external ophthalmoplegia and two had limb weakness alone. Even within the MELAS subgroup, the majority of patients had one or more clinical manifestations considered to be atypical of the MELAS syndrome. They included developmental delay, ophthalmoparesis, pigmentary retinopathy and intestinal pseudo-obstruction. The proportion of mutant mitochondrial DNA (mtDNA) in muscle was generally higher in patients with recurrent strokes than in those without strokes, the highest levels being observed in MELAS cases with early onset disease. Studies of isolated muscle mitochondria identified a range of respiratory chain abnormalities mostly involving Complex I; immunoblots of Complex I in 3 of 10 cases showed selective loss of specific subunits encoded by nuclear genes. In the group as a whole, however, no clear correlations were observed between the severity or extent of the respiratory chain abnormality and clinical phenotype or the proportion of mutant mtDNA in biopsied skeletal muscle. These discrepancies suggest that, in patients harbouring the common MELAS3243 mutation, differences in heteroplasmy and the proportions of mutant mtDNA may not be the sole determinants of disease expression and that additional genetic mechanisms are involved in defining the range of clinical and biochemical phenotypes associated with this aberrant mitochondrial genome.

The molecular pathology of respiratory-chain dysfunction in human mitochondrial myopathies.

Some of the different molecular pathologies of respiratory-chain dysfunction in human mitochondrial myopathies will be reviewed in relation to the findings in 58 cases. Deletions of mitochondrial DNA were identified in 21 cases [36%]. There was some correlation between the sites of the deletion and the mitochondrial biochemistry in patients with defects of Complex I but not in cases with more extensive loss of respiratory chain activity. Complex I and Complex IV polypeptides were usually normal in deleted cases. Non-deleted cases, however, often showed specific subunit deficiencies which involved the products of both nuclear and mitochondrial genes. Immunoblots of respiratory-chain polypeptides in one case pointed to defective translocation of the Rieske precursor from the cytosol into the mitochondria. The pathogenic role of circulating autoantibodies to specific matrix proteins and the nature of the target antigens in two patients with mitochondrial encephalomyopathies and respiratory-chain dysfunction will also be discussed.

New phenotypic diversity associated with the mitochondrial tRNA(SerUCN) gene mutation.

We performed detailed clinical, histopathological, biochemical, in vitro translation and molecular genetic analysis in patients from two unrelated families harbouring the tRNA(SerUCN) 7472C-insertion mutation. Proband 1 developed a progressive neurodegenerative phenotype characterised by myoclonus, epilepsy, cerebellar ataxia and progressive hearing loss. Proband 2 had a comparatively benign phenotype characterised by isolated myopathy with exercise intolerance. Both patients had the 7472C-insertion mutation in identical proportions and they exhibited a similar muscle biochemical and histopathological phenotype. However, proband 2 also had a previously unreported homoplasmic A to C transition at nucleotide position 7472 in the tRNA(SerUCN) gene. This change lengthens further the homopolymeric C run already expanded by the 7472C-insertion. These data extend the phenotypic range associated with the 7472C-insertion to include isolated skeletal myopathy, as well as a MERRF-like phenotype.

The neuropsychological features of mitochondrial myopathies and encephalomyopathies.

Detailed testing of higher cerebral function was performed in 36 patients with mitochondrial myopathies and encephalomyopathies. Fourteen of these patients were thought to be cognitively impaired on clinical grounds. The assessments included tests of general intellectual ability and focal tests of memory, language, and perception. Twenty-one (58%) of the 36 patients who were tested had evidence of general intellectual deterioration, with focal cognitive deficits of variable degree. Of the remaining 15 patients in whom there was no evidence of general intellectual decline, five displayed focal cognitive deficits. In only 10 patients was there evidence of cerebral dysfunction. The range and extent of cognitive deficits in mitochondrial myopathies are greater than predicted by their clinical presentations.

A novel mutation in the mitochondrial tRNA(TYr) gene associated with exercise intolerance.

The authors report a novel A5874G mutation in the mitochondrial tRNA tyrosine (tRNA(TYr)) gene associated with exercise intolerance, limb weakness, and complex III deficiency. The mutation was absent in blood from the patient and all maternal family members, indicating that it may be a spontaneous somatic mutation in muscle. This is the first point mutation in the tRNA(TYr) gene associated with human disease and is further evidence that exercise intolerance associated with complex III deficiency is genetically heterogeneous.

Acanthocytosis, retinitis pigmentosa, and pallidal degeneration: a report of three patients, including the second reported case with hypoprebetalipoproteinemia (HARP syndrome).

We describe an example of a variant of Hallervorden-Spatz disease, characterized by hypoprebeta-lipoproteinemia, acanthocytosis, retinitis pigmentosa, and pallidal degeneration (HARP syndrome), in an 18-year-old woman who presented with longstanding intellectual subnormality, night blindness, and a 2-year history of orobuccolingual dystonia causing dysarthria and dysphagia. Investigation showed acanthocytosis and hypoprebetalipoproteinemia, and electroretinograms were typical of tapetoretinal degeneration. T2-weighted MRI showed decreased signal intensity in the pallidal nuclei with central hyperintensity, constituting the "eye-of-the-tiger" sign. The patient's sister and mother have a similar lipid disorder but no retinal or neurologic disease. We also report two patients with clinical and radiologic features similar to those of the patient with HARP syndrome but who had normal lipid studies. These various combinations of components of HARP syndrome may be caused by several distinct genetic diseases or may represent variable manifestations of a contiguous gene defect.

Fifty year follow-up of a patient with central core disease shows slow but definite progression.

The follow-up of a patient with central core disease (CCD) over 50 years showed that although initially the condition was moderately non-progressive, progression of a significant degree did eventually occur. Histopathological and electron microscopic data were available from muscle biopsies carried out at the ages of 19 and 55 years, and show a marked predominance of type 1 fibres with central cores in most fibres at both ages. The four mutations within the RYR1 gene described in association with CCD and three of the more common malignant hyperthermia-associated mutations within RYR1 were not present.

Chronic administration of the oral hypoglycaemic agent diphenyleneiodonium to rats. An animal model of impaired oxidative phosphorylation (mitochondrial myopathy).

Daily subcutaneous administration of the oral hypoglycaemic agent, diphenyleneiodonium at a low dose (1.5 mg/kg body weight) over a 4-5 week period resulted in a normoglycaemic stable animal model of impaired oxidative phosphorylation in the rat. Diphenyleneiodonium specifically inhibits NAD-linked mitochondrial oxidation [Bloxham, Biochem. Soc. Trans. 7, 103 (1979)], and in isolated mitochondrial preparations from heart, soleus and gastrocnemius muscle and liver from treated animals NAD-linked respiration was reduced by 40% or more of mean control values. Brain and kidney mitochondria isolated from the treated group had similar rates of NAD-linked respiration to their respective control values. The activity of NADH-ferricyanide reductase was significantly reduced in all tissues tested, even in the isolated brain and kidney mitochondria where the activity in these tissues was 60-75% of control values. This suggests that at least 40% of Complex I activity must be inhibited before there is a decline in NAD-linked mitochondrial respiration. This paper discusses the use of diphenyleneiodonium as a means of establishing an animal model of the human disease state, termed mitochondrial myopathy.

The retinal manifestations of mitochondrial myopathy. A study of 22 cases.

In a series of 61 patients with the morphologic and histochemical features of mitochondrial myopathy, 22 (36%) had pigmentary retinopathy. Three patterns of retinopathy were identified. Eighteen patients had a "salt and pepper" type of retinal appearance, which was usually associated with good visual function. Two had many features of retinitis pigmentosa, and two others showed generalized loss, or atrophy, of the retinal pigment epithelium and choriocapillaris. These last four patients had markedly reduced visual acuities, with optic atrophy and attenuated retinal vessels. Electroretinography and electro-oculography were performed in 11 patients. Both rod and cone mediated electroretinographic functions were subnormal in eight patients, while only cone mediated functions were depressed in the remaining three. The electro-oculographic changes were variable.

Becker muscular dystrophy presenting with complete heart block in the sixth decade.

Becker muscular dystrophy may be associated with myocardial abnormalities which are usually diagnosed after the onset of weakness. We present a patient who developed complete heart block 6 years before the onset of muscle weakness which occurred unusually late at the age of 62 years.

Prognosis in adult polymyositis.

The effects of long-term corticosteroid treatment have been assessed in 20 patients with polymyositis. Cases were only accepted if a muscle biopsy showed the characteristic features of inflammation and muscle fibre degeneration. If the muscle disorder was only a minor feature of generalised collagen disease or there was evidence of coexisting neuropathy, the patients were excluded. Over a mean follow-up period of 5 years, 8 patients died and only 4 improved. These results have been compared with those in other series. An attempt has been made to correlate muscle biopsy findings with prognosis, and the reasons for the poor outcome are discussed.

Mitochondrial myopathy. Biochemical studies revealing a deficiency of NADH--cytochrome b reductase activity.

This paper presents biochemical data upon a young male with a mitochondrial myopathy characterised by weakness, severe exercise intolerance, muscle wasting and exercise-induced lactic acidaemia. Two similar cases have been previously documented (Morgan-Hughes et al. 1979). This report more precisely locates the mitochondrial defect. In vitro mitochondrial studies show markedly decreased respiratory rates with all NAD-linked substrates whilst that with flavin-linked succinate is normal. Oxidative phosphorylation is normally coupled. Mitochondrial cytochrome components as determined by low temperature spectroscopy are normal. NADH-ferricyanide reductase and primary dehydrogenase activities are present at levels far in excess of that required to support normal NAD-linked substrate oxidation rates. Intramitochondrial NAD levels are similar to those found in other mammalian muscle. It is proposed therefore that the mitochondrial defect is situated between NADH dehydrogenase and the CoQ--Cytochrome b complex; possibly being a derangement of a non-haem iron sulphur centre.

Myoadenylate deaminase deficiency or not? Observations on two brothers with exercise-induced muscle pain.

This paper describes 2 brothers with increasingly severe exercise-induced muscle pain and stiffness, beginning in adolescence. Histochemical studies showed that myoadenylate deaminase activity was absent in the propositus, but present in his younger brother. Biochemical examination of muscle homogenates confirmed these findings, with enzyme activity approximately 60% of the mean control value in the younger sibling. Red cell adenylate deaminase activity was normal in both cases. The possible relationship between the clinical and biochemical findings in these patients is discussed.

Kearns-Sayre syndrome associated with mitochondrial DNA deletion or duplication: a molecular genetic and pathological study.

The neuropathological findings in 2 patients with Kearns-Sayre syndrome and mitochondrial DNA (mtDNA) rearrangements, one a predominant deletion and the other a predominant duplication, were remarkably similar, showing diffuse vacuolation of white matter. There were some of the pathological features of Leigh's syndrome in the spinal cord of the patient with a duplication. In the patient with a predominant deletion, rearranged mtDNA was undetectable in blood, spleen, and testis, and present in highest amounts in muscle and the brain, but relatively low in cerebellum, reflecting the ratio seen, albeit in much smaller amounts, in normal aged brains. MtDNA rearrangements in this patient were largely deletions or deletion dimers; duplicated mtDNA was present in only trace amounts in some tissues and there was none in skeletal muscle. The patient with a predominant duplication of mtDNA had higher amounts of rearranged mtDNA in blood (mainly duplicated) than muscle (mainly deleted). Correlation of these data with tissue dysfunction is probably complicated by the replicative behaviour of deleted, duplicated and normal mtDNA.

Impaired mitochondrial translation in human myoblasts harbouring the mitochondrial DNA tRNA lysine 8344 A-->G (MERRF) mutation: relationship to proportion of mutant mitochondrial DNA.

The mitochondrial DNA transfer RNA lysine A8344G mutation is commonly associated with the MERRF (myoclonus epilepsy with ragged red fibre) phenotype. The molecular pathogenesis of disease associated with this mutation is unclear. Theoretically, a mitochondrial tRNA mutation might affect transcription or translation, or both. We therefore studied these processes in cloned primary human myoblast cultures containing different proportions of mutant mtDNA. No abnormality of transcription was observed. However, there was a progressive decrease in mitochondrially encoded protein synthesis as the proportion of mutant mtDNA increased. Furthermore, there was evidence that subunits were differentially affected, based on selective reduction of cytochrome c oxidase subunits with relatively low proportions of mutant mtDNA.

A mitochondrial myopathy with a deficiency of respiratory chain NADH-CoQ reductase activity.

This paper presents data on two sisters with a mitochondrial myopathy characterised by weakness, marked exercise intolerance and a fluctuating lactic acidaemia. Both patients also experienced episodes of increased weakness which could be brought on by unaccustomed activity, going without food or by taking small quantities of alcohol. Metabolic studies during exercise showed a marked and sudden rise in blood lactate and pyruvate levels. Biochemical studies in one case showed that mitochondrial respiratory rates were markedly decreased with all NAD-linked substrates tested but were normal with succinate and with TMPD + ascorbate. The mitochondrial cytochrome components were normal as determined by low temperature spectroscopy and the addition of uncoupler did not enhance state 3 respiratory rates utilising NAD-linked substrates. It was concluded, therefore, that the mitochondrial lesion was located at the level of the NADH-CoQ reductase complex.

Prolonged aerobic exercise: physiological studies in rat gastrocnemius with additional observations on the effects of acute mitochondrial blockade.

Protracted low frequency (1 and 5 Hz) stimulation of rat gastrocnemius in vivo leads to fatigue and fall out of glycolytic fibres with the development of a stable twitch response with continuation of the stimulus train. This model was used to examine the physiological effects of acute mitochondrial blockade on oxidative fibre function with the injection of a mitochondrial uncoupler (dinitrophenol) and a site I inhibitor (diphenyleneiodonium) intraarterially after a stable twitch response had developed. Dinitrophenol leads to progressive failure of contractility, closely followed by action potential failure and electrically silent contracture: external work accelerated this sequence but it also developed in resting muscle suggesting that DNP lead to active ATP hydrolysis and a more severe energy depletion than that encountered in human disease states. Diphenyleneiodonium also leads to progressive twitch tension and action potential failure but contracture was late and inconstant, considerable recovery in twitch parameters was seen with rest and restimulation lead to pathological fatiguability of twitch tension. This model has some similarity to human mitochondriopathies with pathological fatiguability. This acute model should allow ready testing of any therapeutic approaches which bypass respiratory chain blocks.

Affinity of antigen-specific IgG distinguishes multiple sclerosis from encephalitis.

The characteristics of antigen-specific IgG in patients with multiple sclerosis and patients with encephalitis have been compared. Both groups of patients showed antigen-specific oligoclonal bands locally synthesised in the CSF. When the affinity distribution of the antigen-specific IgG was measured there was a marked difference between the two groups. Encephalitis patients had high affinity antibody against the causative antigen. This was consistent with the antibody undergoing affinity maturation as a result of the immune system fighting a primary infection. Multiple sclerosis patients lacked high affinity response. This lack of high affinity antibody was also seen in those encephalitis patients when antigens other than the causative antigen were studied.