Contrasting phenotypes in three patients with novel mutations in mitochondrial tRNA genes

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Abstract

We studied three patients, each harboring a novel mutation at a highly conserved position in a different mitochondrial tRNA gene. The mutation in patient 1 (T5543C) was associated with isolated mitochondrial myopathy, and occurred in the anticodon loop of tRNATrp. In patient 2, with mitochondrial myopathy and marked retinopathy, the mutation (G14710A) resulted in an anticodon swap (Glu to Lys) in tRNAGlu. Patient 3, who manifested mitochondrial encephalomyopathy and moderate retinal dysfunction, harbored a mutation (C3287A) in the TψC loop of tRNALeu(UUR). The mutations were heteroplasmic in muscle in all cases, and sporadic in two cases. PCR-RFLP analysis in all patients showed much higher amounts of mutated mtDNA in affected tissue (muscle) than unaffected tissue (blood), and significantly higher levels of mutated mtDNA in cytochrome c oxidase (COX)-negative muscle fibers than in COX-positive fibers, confirming the pathogenicity of these mutations. The mutation was also detected in single hair roots from all three patients, indicating that each mutation must have arisen early in embryonic development or in maternal germ cells. This suggests that individual hair root analyses may reflect a wider tissue distribution of mutated mtDNA than is clinically apparent, and might be useful in predicting prognosis and, perhaps, the risk of transmitting the mutation to offspring. Our data suggest a correlation between clinical phenotype and distribution of mutated mtDNA in muscle versus hair roots. Furthermore, the high threshold for phenotypic expression in single muscle fibers (92–96%) suggests that therapies may only need to increase the percentage of wild-type mtDNA by a small amount to be beneficial.

Introduction

The mitochondrial myopathies and encephalomyopathies (MIM251900) encompass a diverse group of disorders of mitochondrial function, frequently associated with defects of the respiratory chain [1], [2]. The diversity of these disorders reflects not only the complexity of the respiratory chain itself, made up of >80 individual proteins that are organized into five major complexes (I–V), but also the unique properties of mtDNA which encodes 13 of these polypeptides: 7 (ND1-6 and 4L) in complex I (NADH:ubiquinone oxidoreductase; EC 1.6.5.3.); 1 (cytochrome b) in complex III (ubiquinol:cytochrome c reductase; EC 1.10.2.2.); 3 (COX I-III) in complex IV (cytochrome c oxidase; COX; EC 1.9.3.1.); and 2 (A6 and A8) in complex V (ATP synthase; EC 3.6.1.34.). MtDNA also encodes 2 rRNA genes and 22 tRNA genes. Because of the small size and known sequence of human mtDNA [3], many mutations have now been recognized [4], [5]. Hundreds of large-scale rearrangements of mtDNA and over 60 pathogenic mutations in tRNA genes have been reported [6]. These are now recognized as a major cause of neuromuscular disease. Pathogenic mutations in the rRNA or protein-coding genes are less frequent. Most disorders of mtDNA are heteroplasmic, meaning that both normal and mutated mtDNA species are present. They are frequently associated with so-called ragged red fibers (RRF) seen on muscle biopsies, reflecting the proliferation of mitochondria containing pathogenic mtDNA mutations in energy-deficient fibers. In contrast to the major rearrangements of mtDNA, which are usually sporadic, many mutations in tRNA, rRNA or protein-coding genes are maternally inherited. Well known examples are the A3243G mutation in the mitochondrial tRNALeu(UUR) gene which is associated with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS [MIM 540000]) and the A8344G mutation in the tRNALys gene, which is associated with myoclonic epilepsy with RRF (MERRF [MIM 545000]). These disorders also manifest clinical and genetic heterogeneity, likely contributing to their under-recognition. For example, families harboring the A3243G mutation can present with either MELAS, PEO (progressive external ophthalmoplegia), or with isolated diabetes and deafness [1]. On the other hand, several other mutations of mtDNA can also produce the MELAS phenotype. Variability of expression among, and even within, different families may reflect the load of mutated mtDNA, its distribution in different tissues, and the influence of nuclear-encoded genes [7], [8], [9]. All of this contributes to the complexity of diagnosing these diseases and providing accurate prognostic and genetic counseling to affected families.

Many patients with tRNA mutations have a complex multisystem disease which may include neurological or psychiatric symptoms, pigmentary retinopathy, sensorineural hearing loss, myopathy, cardiomyopathy, gastrointestinal symptoms or diabetes. However, rare cases manifest isolated myopathy or cardiomyopathy. Although the majority of mutations of mtDNA-encoded tRNA genes are maternally inherited, sporadic cases have been described [10], [11], [12].

With the goal of increasing our knowledge of the phenotypic expression and pathogenesis of these disorders, we now describe three patients whose clinical presentations vary from a predominant myopathy to an encephalomyopathy with multisystem involvement. In each patient, we have identified a novel mutation in an mtDNA-encoded tRNA gene, and we present evidence for the pathogenicity of these mutations. Furthermore, we have demonstrated that investigation of mutated and wild-type mtDNA in tissues of different embryonic origin can provide both diagnostic and potentially prognostic information, as well as information on the origin of these mutations. Preliminary reports of patient 1 have been published [13], [14].

Section snippets

Patient 1

This 58-year-old male dentist had a normal childhood but led a sedentary lifestyle following a retinal detachment at 11 years of age. He also experienced severe atopic dermatitis since adolescence. He first noted easy fatigability and breathlessness, and elevated blood cholesterol, in his late 30s. He was referred to a cardiologist who performed a stress test and prescribed a low cholesterol, weight-loss diet, and an exercise program which he could never complete, and which left him exhausted.

Identification of a unique mtDNA-encoded tRNA gene mutation in each patient

The clinical histories, the presence of RRF and COX-negative fibers on muscle histochemistry, and the finding of low respiratory chain activities in muscle mitochondria, suggested the possibility of an mtDNA mutation in each of our patients. Furthermore, Southern blot analysis ruled out the presence of a major rearrangement of mtDNA. Therefore, sequencing of all mtDNA-encoded tRNA genes was performed (Table 1). This revealed a unique, novel tRNA gene mutation in each patient: a T5543C

Discussion

The diagnosis of a mitochondrial myopathy, retinopathy or encephalomyopathy in our three patients was based on the clinical phenotype, visual electrophysiology, the identification of RRF and COX-negative fibers on muscle histochemistry, and the reduction of one or more respiratory chain activities in muscle mitochondria. This suggested the possibility of a mutation of mtDNA and led to the identification of a novel and unique mtDNA-encoded tRNA mutation in each patient. Evidence for the

Electronic-database information

URLs for data in this article are as follows: Online Mendelian Inheritance in Man (OMIM), http://www3.ncbi.nlm.nih.gov/Omim (for complex I deficiency, complex III deficiency, complex IV (COX) deficiency, complex V deficiency, MELAS, MERRF, mitochondrial myopathy, and tRNAGlu, tRNALeu(UUR), tRNALeu(CUN), tRNAPro, tRNATrp, and tRNATyr mutations).

Acknowledgments

We acknowledge the following individuals who contributed to the clinical evaluation, work-up and care of these patients: Drs. B. Powell, D. Elliot, R.H. Haller, W. Johnston, T. Taivassalo, E.A. Zimmerman, and Dr. A. Penn (for 31P MRS). We also thank Drs. K.M. Gibson, B. Popovich and their staff for sharing laboratory space and providing technical advice, and Beth Lee for measurement of respiratory chain activities. This work was supported by grants from the Muscular Dystrophy Association (to

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    Present address: Australian Centre for Astrobiology, Macquarie University, North Ryde, 2109, NSW, Australia.

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