Martin J. Barron

The diagnosis of mitochondrial muscle disease

Mitochondrial respiratory chain abnormalities are an important cause of neuromuscular disease and may be due to defects of either the mitochondrial or nuclear genome. On account of the clinical and genetic heterogeneity exhibited by the mitochondrial myopathies, their investigation and diagnosis remains a challenge, requiring a combination of techniques including muscle histochemistry, biochemical assessment of respiratory chain function and molecular genetic studies. Here, we describe a step-by-step approach to the clinical and laboratory diagnosis of mitochondrial muscle disease, highlighting the many potential problems that can hinder reaching the correct diagnosis.

A homoplasmic mitochondrial transfer ribonucleic acid mutation as a cause of maternally inherited hypertrophic cardiomyopathy

OBJECTIVES The purpose of this study was to understand the clinical and molecular features of familial hypertrophic cardiomyopathy (HCM) in which a mitochondrial abnormality was strongly suspected. BACKGROUND Defects of the mitochondrial genome are responsible for a heterogeneous group of clinical disorders, including cardiomyopathy. The majority of pathogenic mutations are heteroplasmic, with mutated and wild-type mitochondrial deoxyribonucleic acid (mtDNA) coexisting within the same cell. Homoplasmic mutations (present in every copy of the genome within the cell) present a difficult challenge in terms of diagnosis and assigning pathogenicity, as human mtDNA is highly polymorphic. METHODS A detailed clinical, histochemical, biochemical, and molecular genetic analysis was performed on two families with HCM to investigate the underlying mitochondrial defect. RESULTS Cardiac tissue from an affected child in the presenting family exhibited severe deficiencies of mitochondrial respiratory chain enzymes, whereas histochemical and biochemical studies of the skeletal muscle were normal. Mitochondrial DNA sequencing revealed an A4300G transition in the mitochondrial transfer ribonucleic acid (tRNA)(Ile) gene, which was shown to be homoplasmic by polymerase chain reaction/restriction fragment length polymorphism analysis in all samples from affected individuals and other maternal relatives. In a second family, previously reported as heteroplasmic for this base substitution, the mutation has subsequently been shown to be homoplasmic. The pathogenic role for this mutation was confirmed by high-resolution Northern blot analysis of heart tissue from both families, revealing very low steady-state levels of the mature mitochondrial tRNA(Ile). CONCLUSIONS This report documents, for the first time, that a homoplasmic mitochondrial tRNA mutation may cause maternally inherited HCM. It highlights the significant contribution that homoplasmic mitochondrial tRNA substitutions may play in the development of cardiac disease. A restriction of the biochemical defect to the affected tissue has important implications for the screening of patients with cardiomyopathy for mitochondrial disease.

The distributions of mitochondria and sodium channels reflect the specific energy requirements and conduction properties of the human optic nerve head

Aim: To study the normal distributions of mitochondria and voltage gated Na+ channels in the human optic nerve head in order to gain insight into the potential mechanisms of optic nerve dysfunction seen in the inherited optic neuropathies. Methods: Five fresh frozen human optic nerves were studied. Longitudinally orientated, serial cryosections of optic nerve head were cut for mitochondrial enzyme histochemistry and immunolabelling for cytochrome c oxidase (COX) subunits and voltage gated Na+ channel subtypes (Nav 1.1, 1.2, 1.3, and 1.6). Results: A high density of voltage gated Na+ channels (subtypes Nav 1.1, 1.3, and 1.6) in the unmyelinated, prelaminar, and laminar optic nerve was found. This distribution co-localised both with areas of high COX activity and strong immunolabelling for COX subunits I and IV. Conclusions: Increased numbers of mitochondria in the prelaminar optic nerve have previously been interpreted as indicating a mechanical hold up of axoplasmic flow at the lamina cribrosa. These results suggest that this increased mitochondrial density serves the higher energy requirements for electrical conduction in unmyelinated axons in the prelaminar and laminar optic nerve and is not a reflection of any mechanical restriction. This could explain why optic neuropathies typically occur in primary inherited mitochondrial diseases such as Leber’s hereditary optic neuropathy, myoclonic epilepsy with ragged red fibres (MERRF), and Leigh’s syndrome. Secondary mitochondrial dysfunction has also been reported in dominant optic atrophy, Friedreich’s ataxia, tobacco alcohol amblyopia, Cuban epidemic optic neuropathy, and chloramphenicol optic neuropathy. These diseases are rare but these findings challenge the traditional theories of optic nerve structure and function and may suggest an alternative approach to the study of commoner optic neuropathies such as glaucoma.

Familial myopathy: New insights into the T14709C mitochondrial tRNA mutation

We have defined the genetic defect in a large family first described in one of the earliest reports of suspected mitochondrial myopathy, as the mutation T14709C in the mitochondrial transfer RNAGlu (mt‐tRNAGlu) gene. Extraordinarily, this mutation has attained homoplasmy (100% mutated mt‐tRNAGlu) on at least three independent occasions in this family and has done so in one individual who remains asymptomatic with no clinical evidence of disease. Heteroplasmy (dual populations of mutated and wild‐type mtDNA) usually is regarded as one of the primary diagnostic criteria for pathogenicity and previous reports of the T14709C mutation detail heteroplasmy in a variety of tissues. In contrast, homoplasmy of mt‐tRNA mutations generally has been regarded as evidence of a benign nature, with rare exceptions that result in organ‐specific phenotypes. Discovering that T14709C, a common and severe mt‐tRNA mutation, can attain homoplasmy without symptoms or clinical signs of disease has profound implications for the identification and prevalence of other pathogenic mt‐tRNA mutations. Furthermore, variation in phenotype between homoplasmic individuals implies a crucial contribution from the nuclear genetic environment in determining the clinical outcome of mt‐tRNA mutations. Ann Neurol 2004;55:000–000

Mitochondrial DNA Defects and Selective Extraocular Muscle Involvement in CPEO

PURPOSE. Chronic progressive external ophthalmoplegia (CPEO) is a prominent, and often the only, presentation among patients with mitochondrial diseases. The mechanisms underlying the preferential involvement of extraocular muscles (EOMs) in CPEO were explored in a comprehensive histologic and molecular genetic study, to define the extent of mitochondrial dysfunction in EOMs compared with that in skeletal muscle from the same patient. METHODS. A well-characterized cohort of 13 CPEO patients harboring a variety of primary and secondary mitochondrial (mt)DNA defects was studied. Mitochondrial enzyme function was determined in EOM and quadriceps muscle sections with cytochrome c oxidase (COX)/succinate dehydrogenase (SDH) histochemistry, and the mutation load in single muscle fibers was quantified by real-time PCR and PCR-RFLP assays. RESULTS. CPEO patients with mtDNA deletions had more COX-deficient fibers in EOM (41.6%) than in skeletal muscle (13.7%, P > 0.0001), and single-fiber analysis revealed a lower mutational threshold for COX deficiency in EOM. Patients with mtDNA point mutations had a less severe ocular phenotype, and there was no significant difference in the absolute level of COX deficiency or mutational threshold between these two muscle groups. CONCLUSIONS. The more pronounced mitochondrial biochemical defect and lower mutational threshold in EOM compared with skeletal muscle fibers provide an explanation of the selective muscle involvement in CPEO. The data also suggest that tissue-specific mechanisms are involved in the clonal expansion and expression of secondary mtDNA deletions in CPEO patients with nuclear genetic defects.

Defects in multiple complexes of the respiratory chain are present in ageing human colonic crypts

Mitochondrial DNA (mtDNA) mutations accumulate in a number of ageing tissues and are proposed to play a role in the ageing process. We have previously shown that colonic crypt stem cells accumulate somatic mtDNA point mutations during ageing. These mtDNA mutations result in the loss of the activity of complex IV (cytochrome c oxidase (COX)) of the respiratory chain in the stem cells and their progeny, producing colonic crypts which are entirely COX deficient. However it is not known whether the other complexes of the respiratory chain are similarly affected during ageing. Here we have used antibodies to individual subunits of complexes I–IV to investigate their expression in the colonic epithelium from human subjects aged 18–84. We show that in ∼50% of crypts with any form of respiratory chain deficiency, decreased expression of subunits of multiple complexes is observed. Furthermore we have sequenced the entire mitochondrial genome of a number of cells with multiple complex defects and have found a wide variety of point mutations in these cells affecting a number of different protein encoding and RNA encoding genes. Finally we discuss the possible mechanisms by which multiple respiratory chain complex defects may occur in these cells.

Differences in the accumulation of mitochondrial defects with age in mice and humans.

Mitochondrial DNA mutations and associated defects in cytochrome c oxidase (COX) are proposed to play an important role in human ageing; however there have been limited studies on the frequency of these defects in normal mouse ageing. Here we compare COX-deficiency in two epithelial tissues; the colon and the ciliary epithelium, from human and mouse. The pattern of accumulation of COX-deficiency is similar in both tissues in the two species; however the frequency of colonic crypts with COX-deficiency in aged humans is significantly higher than in aged mice, whereas the levels of COX-deficiency in the ciliary epithelium are higher in the mouse than in humans. This suggests the impact of mitochondrial defects on normal ageing may differ significantly between species.

GASTROINTESTINAL TRACT INVOLVEMENT ASSOCIATED WITH THE 3243A>G MITOCHONDRIAL DNA MUTATION

Defects of the mitochondrial genome (mtDNA) are increasingly recognized as common causes of neurologic syndromes.1 One of the most common pathogenic mtDNA mutations is the m.3243A>G in the MTTL1 gene. In addition to numerous neurologic features this mutation can also give rise to gastrointestinal symptoms including bloating, dysphagia, recurrent vomiting and anorexia, chronic diarrhea, and gastrointestinal pseudo-obstruction.2–4 To gain insight into the pathophysiology of gastrointestinal symptoms associated with the m.3243A>G mtDNA mutation, we investigated the degree of respiratory chain deficiency and the level of m.3243A>G mutation in individual areas from the gastrointestinal tract of two patients in whom gastrointestinal symptoms were both prominent and difficult to manage. ### Case report. Patient 1 developed many features of the mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS) syndrome including strokelike episodes, encephalopathy, myopathy, and lactic acidosis. In addition, she had a long history of digestive problems dating back to childhood. She had a small appetite even as a child and felt full even with small portions. During her teenage years constipation became more marked, as well as a feeling of bloating after even a small meal. In later life she developed severe constipation requiring regular enemas and laxatives. She died at age 32 years. Patient 2, who died at age 59 years, was the maternal aunt of Patient 1. She had a …

Cytochrome c oxidase deficient muscle fibres: Substantial variation in their proportions within skeletal muscles from patients with mitochondrial myopathy

Mitochondrial DNA (mtDNA) disease is a common cause of myopathy and the presence of histochemically demonstrated cytochrome c oxidase (COX) deficiency is an extremely useful diagnostic feature. However, there is currently no quantitative information regarding the variability of COX deficiency within or between muscles. This study addresses this issue by studying a number of skeletal muscle samples obtained at post-mortem from three patients with mitochondrial disease due to established mitochondrial DNA defects. COX deficient muscle fibres were enumerated in sections of these muscles and analysed according to patient, individual muscle, position within a particular muscle and sample size. Descriptive statistics were generated followed by an analysis of variance (ANOVA) to assess the effect of these parameters on the mean percentage of COX deficient fibres. We observed statistically significant variation in the percentage of COX deficient fibres within individual muscles from each patient for samples sizes of between 100 and 400 fibres. Our results have implications for the way in which biopsies of skeletal muscle are used for the assessment of disease severity, progression and response to treatment.