A late-onset mitochondrial myopathy is associated with a novel mitochondrial DNA (mtDNA) point mutation in the tRNATrp gene

doi:10.1016/S0960-8966(98)00037-6

Neuromuscular Disorders

Volume 8, Issue 5, June 1998, Pages 291-295

https://doi.org/10.1016/S0960-8966(98)00037-6 Get rights and content

Abstract

We detected a novel pathogenic mutation, a G→A transition at position 5521 of mitochondrial tRNA^Trp gene, in association with familial late-onset mitochondrial myopathy. The mutation was detected in muscle but not in leukocytes from the family's proband. Morphological and biochemical studies documented a severe defect of muscle cytochrome c oxidase (COX) activity. RFLP analysis of single muscle fibers demonstrated segregation of higher percentages of mutated genomes in COX-negative ragged red fibres compared with normal fibers. A predominant impairment in synthesis of subunits I and III of complex IV due to their highest relative content of tryptophane might explain the greater susceptibility of complex IV to the pathogenic effect of this mutation. A progressive accumulation of mutated genomes in muscle can account for the late onset of symptoms observed in affected members.

Introduction

Mitochondrial diseases due to mitochondrial tRNA gene mutations are usually multisystem disorders with infantile or juvenile onset of symptoms. Late onset rarely exceeds middle-age and is seldom reported in full-syndrome patients. It can, however, be more commonly observed in oligosymptomatic relatives.

The biochemical phenotype expressed by a tRNA mutation is usually characterized by a partial generalized decrease of all respiratory chain enzymes containing mtDNA-encoded subunits, which reflects the generalized impairment of mtDNA translation caused by tRNA malfunction.

We now report a novel mtDNA point mutation located in the tRNA^Trp gene associated with a familial mitochondrial myopathy characterized by peculiar clinical and biochemical features. All affected members had a late onset of symptoms; biochemical studies on the proband's muscle showed a selective decrease of cytochrome c oxidase (COX) activity, while other respiratory chain activities were in the normal range.

Section snippets

Materials and methods

Histochemistry and biochemistry on muscle, DNA extraction, Southern blot and analysis for MELAS 3243/3271, MERRF 8344/8356 and PEO 4285/5703/5692 mutations and the sequence of tRNA genes were done as described [1]. Single-fiber PCR was performed according to Moraes and Schon [2].

Screening for the G→A at nt. 5521 of mtDNA was done by RFLP analysis of a 91 bp PCR fragment amplified by modified primers. We used a reverse oligonucleotide (nt. 5550–5580 according to Anderson's sequence [3])

Case report and results

A 68-year-old male noticed progressive bilateral ptosis and fatigue since the age of 50. His mother, deceased at age 90, and one of his brothers were reported to be similarly affected (the pedigree is represented in Fig. 3). Clinical examination documented bilateral ptosis without ophthalmoplegia, dysphonia and mild proximal muscle wasting and weakness (F=4, MRC scale).

CK and LDH were normal. EMG was myopathic. Muscle biopsy showed a mitochondrial myopathy with many COX-negative ragged red

Discussion

The G→A transition at nt. 5521 of mitochondrial tRNA^Trp gene fulfills all diagnostic criteria for pathogenic mutations, i.e. (i) it is located in a conserved region of the gene, (ii) it has never been reported as neutral polymorphism, nor found in 110 controls and (iii) RFLP analysis demonstrated a condition of heteroplasmy and single-fiber PCR documented higher amounts of mutated mtDNAs in COX− RRFs compared with normal fibers.

There was a good correlation between clinical phenotype,

Acknowledgements

We thank Enrico Paris and Manuela Papacci for their precious technical assistance. This work was supported by UILDM sez. Laziale, Rome and by Telethon Italy (grant n.52).

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In the last 20 years, many mutations affecting mt-tRNAs have been linked to different forms of mitochondrial diseases that variably affect the activities of the MRC complexes, some typically resulting in variable severity of predominant or exclusive COX deficiency, depending on the levels of heteroplasmy. For instance, mutations in mt-tRNALys ([40,41–46,263]), mt-tRNAAla [47–50], mt-tRNAPhe ([51–53]; Almalki et al., 2014), mt-tRNALeu [54–58], mt-tRNATrp [59–66], mt-tRNAAsp [67] cause respiratory defective phenotypes with COX deficiency. But, as seen above, while the MERRF mutation is clearly and consistently associated with COX deficiency, the same is not true for the m.3243 MELAS mutation, which is rather associated with predominant complex I deficiency.
Cytochrome c oxidase (COX) deficiency is characterized by a high degree of genetic and phenotypic heterogeneity, partly reflecting the extreme structural complexity, multiple post-translational modification, variable, tissue-specific composition, and the high number of and intricate connections among the assembly factors of this enzyme. In fact, decreased COX specific activity can manifest with different degrees of severity, affect the whole organism or specific tissues, and develop a wide spectrum of disease natural history, including disease onsets ranging from birth to late adulthood. More than 30 genes have been linked to COX deficiency, but the list is still incomplete and in fact constantly updated. We here discuss the current knowledge about COX in health and disease, focusing on genetic aetiology and link to clinical manifestations. In addition, information concerning either fundamental biological features of the enzymes or biochemical signatures of its defects have been provided by experimental in vivo models, including yeast, fly, mouse and fish, which expanded our knowledge on the functional features and the phenotypical consequences of different forms of COX deficiency.
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We sequenced the mitochondrial genome from a 40-year-old woman with myoclonus epilepsy, retinitis pigmentosa, leukoencephalopathy and cerebral calcifications. Histological and biochemical features of mitochondrial respiratory chain dysfunction were present. Direct sequencing showed a novel heteroplasmic mutation at nucleotide 5513 in the MT-TW gene that encodes tRNA^Trp. Restriction Fragment Length Polymorphism analysis confirmed that about 80% of muscle mtDNA harboured the mutation while it was present in minor percentages in mtDNA from other tissues. The mutation is predicted to disrupt a highly conserved base pair within the aminoacyl acceptor stem of the tRNA. This is the 17° mutation in MT-TW gene and expands the known causes of late-onset mitochondrial diseases.
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Given the etiologic heterogeneity of disease classification using clinical phenomenology, we employed contemporary criteria to classify variants associated with myoclonic epilepsy with ragged-red fibers (MERRF) syndrome and to assess the strength of evidence of gene-disease associations. Standardized approaches are used to clarify the definition of MERRF, which is essential for patient diagnosis, patient classification, and clinical trial design.
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Early onset and severe clinical course associated with the m.5540G>A mutation in MT-TW
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Analysis of the mitochondrial genome from the blood of the patient's mother did not detect the mutation. Mutations in the MT-TW gene coding for the mitochondrial tRNATrp are associated with diverse phenotypes, such as neonatal onset mitochondrial encephalomyopathy [7], mitochondrial myopathy [8], Leigh syndrome [9], dementia and chorea [10], and a neurogastrointestinal syndrome [11]. Here we present the case of a female patient harboring the m.5540G>A transition involving the MT-TW gene.
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A late-onset mitochondrial myopathy is associated with a novel mitochondrial DNA (mtDNA) point mutation in the tRNATrp gene

Abstract

Introduction

Section snippets

Materials and methods

Case report and results

Discussion

Acknowledgements

Biochem Biophys Res Commun

Methods Enzymol

J Neurol Sci

Mol Cell Probes

Sequence and organization of human mitochondrial genome

Nature

A new mitochondrial DNA mutation associated with progressive dementia and chorea: a clinical pathological, and molecular genetic study

Ann Neurol

A late-onset mitochondrial myopathy is associated with a novel mitochondrial DNA (mtDNA) point mutation in the tRNA^Trp gene