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A novel mitochondrial tRNAGlu (MTTE) gene mutation causing chronic progressive external ophthalmoplegia at low levels of heteroplasmy in muscle

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Abstract

Mitochondrial respiratory chain defects are associated with diverse clinical phenotypes in both adults and children, and may be caused by mutations in either nuclear or mitochondrial DNA (mtDNA). We report the molecular genetic investigations of a patient with chronic progressive external ophthalmoplegia (CPEO) and myopathy where muscle biopsies taken 11 years apart revealed a progressive increase in the proportion of cytochrome c oxidase (COX)-deficient fibres. Mitochondrial genetic analysis of the early biopsy had seemingly excluded both mtDNA rearrangements and mtDNA point mutations. Sequencing mtDNA from individual COX-deficient muscle fibres in the second biopsy, however, identified an unreported m.14723 T > C substitution within the mitochondrial tRNAGlu (MTTE) gene, which fulfilled all canonical criteria for pathogenicity. The m.14723 T > C mutation was absent from several tissues, including muscle, from maternal relatives suggesting a de novo event, whilst quantitative analysis of the first muscle biopsy confirmed a very low level of the mutation (7% mutated mtDNA), highlighting a potential problem whereby pathogenic mtDNA mutations may remain undetected using established screening methodologies.

Introduction

Defects of the mitochondrial respiratory chain are associated with a diverse and ever-growing spectrum of clinical phenotypes in both adults and children, and may be caused by mutations in either the nuclear or mitochondrial genome (mtDNA). Where mitochondrial disease is due to mtDNA mutations, those occurring within mitochondrial (mt-) tRNA genes are the most common, with over 150 discrete pathogenic mutations identified across each of the 22 mt-tRNA genes [1]. They are associated with a number of distinctive clinical presentations ranging from the multi-organ, syndromic phenotypes of MERRF and MELAS, to isolated symptoms such as CPEO, diabetes or myopathy. In spite of the clinical and genetic heterogeneity associated with mt-tRNA mutations, determinants of pathogenicity are typically shared across the spectrum [2], [3], [4]. Pathogenic mutations are typically heteroplasmic, a term that describes the presence of both wildtype and mutant mtDNA molecules in a cell. They are present at high levels of heteroplasmy in post-mitotic tissues, such as muscle; are often lost from rapidly dividing cells, such as blood leucocytes [5] and are typically associated with a mosaic pattern of cytochrome c oxidase (COX) deficiency. Their presence at high levels of heteroplasmy in clinically-relevant tissues facilitates molecular diagnosis by direct sequencing of the whole mitochondrial genome.

We present a patient with chronic progressive external ophthalmoplegia (CPEO) and COX-deficient muscle fibres associated with a novel m.14723 T > C mt-tRNAGlu mutation which was present at low levels in the first of two muscle biopsies, thereby evading detection by established sequencing methodologies. The m.14723 T > C mutation clearly segregated with a functional, biochemical defect as measured by COX activity in individual muscle fibres, and the absence of this mutation in muscle and several mitotic tissues from the patient's daughter indicates that it is not transmitted through the female germline.

Section snippets

Case report

The index patient is a 63-year-old female who initially developed bilateral eyelid droop in early childhood, which was evident in photographs taken at the age of five years. She underwent corrective squint surgery at the age of seven years, and has had slight amblyopia in the left eye, having never suffered from significant double vision. She underwent further ophthalmological review at the age of 38 for worsening bilateral ptosis, subsequently requiring surgical correction on two separate

Muscle histology and histochemistry

Standard histological and histochemical analyses of quadriceps muscle biopsy were performed on fresh frozen sections (10 μm). Cytochrome c oxidase (COX) staining was carried out on the patient's initial muscle biopsy whilst investigations on the subsequent patient biopsy included the determination of sequential COX/succinate dehydrogenase (SDH) activities using standard methods [6].

Molecular genetic studies

Total DNA was extracted from several tissues including blood, buccal and urinary epithelium and skeletal muscle

Histology and histochemistry

COX histochemistry of the initial muscle biopsy taken in 1997 revealed 1–2% COX-deficient fibres (Fig. 1A), which first prompted the investigation of this patient. Analysis of the subsequent biopsy, taken 11 years later, demonstrated a significant increase in the proportion of COX-deficient fibres (7% of total biopsy) (Fig. 1B). Sequential COX-SDH analysis revealed that many of the COX-deficient fibres also exhibited subsarcolemmal mitochondrial aggregates which would correspond to ragged-red

Discussion

We describe a patient who presented with CPEO and muscle biopsy changes suggestive of a mtDNA defect, in whom a novel m.14723 T > C mitochondrial tRNA mutation was identified. This novel substitution was present at an uncharacteristically low level of mtDNA heteroplasmy in an initial muscle biopsy, so low in fact that it was missed by the whole mtDNA genome analysis of muscle homogenate DNA, the established diagnostic screen for the detection of rare or novel mtDNA point mutations. This patient's

Acknowledgements

DMT and RWT acknowledge the continuing financial support of a Wellcome Trust Programme Grant (074454/Z/04/Z) and the UK National Commissioning Group which funds the “Rare Mitochondrial Disorders of Adults and Children” Diagnostic Service (http://www.mitochondrialncg.nhs.uk/index.html).

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