Threshold expression of the tRNALys A8344G mutation in single muscle fibres

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Abstract

We investigated the distribution in skeletal muscle of mitochondrial DNA (mtDNA) with the tRNALys A8344G mutation, which is associated with myoclonus epilepsy and ragged red fibres (MERRF) syndrome. Isolated muscle fibre segments (n=144) from six individuals of two different families carrying the mutation were studied. Two of these individuals were affected by MERRF while four had no or minor clinical symptoms. In one individual with a low overall level of mutated mtDNA (mean 18%) the variation in the proportion of mutated mtDNA between individual muscle fibres ranged from 0 to 80%. This result demonstrates that segregation of the tRNALys A8344G mutation within a tissue may lead to very marked variation of the level of mutated mtDNA between individual cells. There was a very high apparent threshold level of mutated mtDNA (95.3–97.7%) for expression of histochemical cytochrome c oxidase (COX) deficiency in individual muscle fibres. The results indicated that this apparent threshold level varied slightly between patients. Ultrastructural examination revealed that an appreciable proportion of the mitochondria in COX-positive muscle fibres lacked COX activity. Variation in intercellular and interorganellar distribution of mutated mtDNA in addition to the absolute mtDNA copy number may explain differences in clinical phenotypes in patients with high levels of the tRNALys A8344G mutation.

Introduction

Mitochondrial DNA (mtDNA) mutations are an important cause of multisystem disorders [1]. The tRNALys A8344G mutation of mtDNA, which was originally associated with the myoclonus epilepsy and ragged red fibre (MERRF) syndrome [2], has now been linked to many other clinical syndromes, such as Leigh's syndrome, progressive external ophthalmoplegia, stroke-like episodes, multiple lipomas and cardiomyopathy 3, 4, 5, 6, 7. There is always heteroplasmy with a mixture of mutated and wild-type mtDNA. The mutation has been considered functionally recessive since affected individuals show high proportions of mutated mtDNA, while asymptomatic carriers usually show lower levels 3, 5, 8, 9. Although a threshold level of mutated mtDNA for clinical expression has been considered, it has not been clarified if the proportion of mutated mtDNA in a tissue is the only major factor determining if the mutation is expressed. The distribution of mutated mtDNA within a tissue, i.e. intercellular variability, and other factors may influence the phenotypic expression.

In this study we have investigated the distribution and intercellular variability of the tRNALys A8344G mutation within a tissue. We have also studied the apparent threshold for enzyme histochemical expression of the mutation at the single cell level in six individuals from two families carrying the mutation.

Section snippets

Patients

Six members of two unrelated families showing segregation of the tRNALys A8344G mutation were investigated. The pedigrees are presented in Fig. 1. A summary of the clinical findings and references to previous publications including these patients are presented in Table 1. Muscle specimens were obtained by open biopsy. The specimens were frozen in a mixture of propane and propylene chilled by liquid nitrogen and stored at −70°C until analyzed.

Enzyme histochemical staining and isolation of single muscle fibres

To identify and isolate muscle fibre segments with

Results

The distribution of mtDNA with the tRNALys A8344G mutation in muscle tissue of six individuals from two unrelated families carrying the mutation was studied by single-fibre PCR analysis. The proportion of mutated and wild-type mtDNA in single muscle fibres is presented in Fig. 2 and Table 2. Fibres with histochemical COX deficiency were present in four of the cases. All COX-deficient muscle fibres showed a high percentage of mutated mtDNA and there was a sharp apparent threshold level for the

Discussion

We have analyzed the apparent threshold level for enzyme histochemical expression of the tRNALys A8344G mutation in single muscle fibres of six patients from two unrelated families carrying the mutation. COX-deficient muscle fibres showed very high levels of mutated mtDNA. In some COX-positive muscle fibres the proportion of mutated mtDNA exceeded 95%, illustrating the functionally recessive nature of this mutation. There was a considerable variation in the proportion of mutated mtDNA in

Acknowledgements

This study was supported by the Swedish Medical Research Council (Proj Nos 07122 and 10823). The valuable help and advice on statistical analysis by Anders Odén, PhD, is gratefully acknowledged.

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