Distribution and threshold expression of the tRNA(Lys) mutation in skeletal muscle of patients with myoclonic epilepsy and ragged-red fibers (MERRF).

We investigated the distribution and expression of mutant mtDNAs carrying the A-to-G mutation at position 8344 in the tRNA(Lys) gene in the skeletal muscle of four patients with myoclonus epilepsy and ragged-red fibers (MERRF). The proportion of mutant genomes was greater than 80% of total mtDNAs in muscle samples of all patients and was associated with a decrease in the activity of cytochrome c oxidase (COX). The vast majority of myoblasts, cloned from the satellite-cell population in the same muscles, were homoplasmic for the mutation. The overall proportion of mutant mtDNAs in this population was similar to that in differentiated muscle, suggesting that the ratio of mutant to wild-type mtDNAs in skeletal muscle is determined either in the ovum or during early development and changes little with age. Translation of all mtDNA-encoded genes was severely depressed in homoplasmic mutant myoblast clones but not in heteroplasmic or wild-type clones. The threshold for biochemical expression of the mutation was determined in heteroplasmic myotubes formed by fusion of different proportions of mutant and wild-type myoblasts. The magnitude of the decrease in translation in myotubes containing mutant mtDNAs was protein specific. Complex I and IV subunits were more affected than complex V subunits, and there was a rough correlation with both protein size and number of lysine residues. Approximately 15% wild-type mtDNAs restored translation and COX activity to near normal levels. These results show that the A-to-G substitution in tRNA(Lys) is a functionally recessive mutation that can be rescued by intraorganellar complementation with a small proportion of wild-type mtDNAs and explain the steep threshold for expression of the MERRF clinical phenotype.     

A tRNA(Lys) mutation in the mtDNA is the causal genetic lesion underlying myoclonic epilepsy and ragged-red fiber (MERRF) syndrome.

Skeletal muscle mtDNA of three patients with mitochondrial encephalomyopathy, characterized clinically by myoclonic epilepsy and ragged-red fiber (MERRF) syndrome, has been sequenced to determine the underlying molecular defect(s). An A-to-G substitution of nt 8344 in the tRNA(Lys) gene, a substitution suggested to be associated with MERRF encephalomyopathy, was detected in these patients. Abnormal patterns of mitochondrial translation products were observed in the skeletal muscle of patients, consistent with the expected consequential defect in protein synthesis. The genealogical studies of the three patients, as well as mtDNA from one published MERRF patient and from nine other normal and disease controls, revealed that the tRNA(Lys) mutations in the MERRF patients have arisen independently. These observations provided evidence that the base substitution is a causal mutation for MERRF.     

Segregation and manifestations of the mtDNA tRNA(Lys) A-->G(8344) mutation of myoclonus epilepsy and ragged-red fibers (MERRF) syndrome.

We have studied the segregation and manifestations of the tRNA(Lys) A-->G(8344) mutation of mtDNA. Three unrelated patients with myoclonus epilepsy and ragged-red fibers (MERRF) syndrome were investigated, along with 30 of their maternal relatives. Mutated mtDNA was not always found in the offspring of women carrying the tRNA(Lys) mutation. Four women had 10%-33% of mutated mtDNA in lymphocytes, and no mutated mtDNA was found in 7 of their 14 investigated children. The presence of mutated mtDNA was excluded at a level of 3:1,000. Five women had a proportion of 43%-73% mutated mtDNA in lymphocytes, and mutated mtDNA was found in all their 12 investigated children. This suggests that the risk for transmission of mutated mtDNA to the offspring increases if high levels are present in the mother and that, above a threshold level of 35%-40%, it is very likely that transmission will occur to all children. The three patients with MERRF syndrome had, in muscle, both 94%-96% mutated mtDNA and biochemical and histochemical evidence of a respiratory-chain dysfunction. Four relatives had a proportion of 61%-92% mutated mtDNA in muscle, and biochemical measurements showed a normal respiratory-chain function in muscle in all cases. These findings suggest that > 92% of mtDNA with the tRNA(Lys) mutation in muscle is required to cause a respiratory-chain dysfunction that can be detected by biochemical methods. There was a positive correlation between the levels of mtDNA with the tRNA(Lys) mutation in lymphocytes and the levels in muscle, in all nine investigated cases. The levels of mutated mtDNA were higher in muscle than in lymphocytes in all cases. In two of the patients with MERRF syndrome, muscle specimens were obtained at different times. In both cases, biochemical measurements revealed a deteriorating respiratory-chain function, and in one case a progressive increase in the amount of cytochrome c oxidase-deficient muscle fibers was found.     

A common mitochondrial DNA mutation in the t-RNA(Lys) of patients with myoclonus epilepsy associated with ragged-red fibers

Nucleotide sequence analyses of muscle mitochondrial DNA (mtDNA) from a patient with myoclonus epilepsy associated with ragged-red fibers (MERRF) revealed 33 single base substitutions, including 23 in coding regions for mitochondrial polypeptides and 10 in non-coding regions, as compared with the normal human mtDNA sequence. Three substitutions, in COI, ND4, and Cytb, would result in amino acid substitutions, which are conserved among species. Of three patients with MERRF, all had an identical A to G base substitution only at nucleotide position 8344 in the t-RNA(Lys) region. The substitution was not found in 15 controls. Various degrees of the combined enzymic defects in the oxidative phosphorylation system of mitochondria were found in the MERRF patients. The defects could be explained by altered function or processing of the mutant t-RNA(Lys). This mutation in the t-RNA(Lys) is the most probable cause of MERRF.     

Multiple symmetric lipomas with high levels of mtDNA with the tRNA(Lys) A-->G(8344) mutation as the only manifestation of disease in a carrier of myoclonus epilepsy and ragged-red fibers (MERRF) syndrome.

We have investigated the morphology, cytogenetics, and the fraction of mtDNA with the tRNA(Lys) A-->G(8344) mutation in three lipomas in a carrier of this mutation. The son of the patient had myoclonus epilepsy and ragged-red fibers syndrome. The fraction of mtDNA with the tRNA(Lys) mutation varied between 62% and 80% in cultured skin fibroblasts, lymphocytes, normal adipose tissue, and muscle. In the three lipomas the mean fraction of mutated mtDNA was 90%, 94%, and 94%. Ultrastructural examination of the lipomas revealed numerous mitochondria with changes such as electron-dense inclusions in some adipocytes. When considered cytogenetically, the lipomas were characterized by a mixture of karyotypically abnormal and normal cells. An identical del(6)(q24) was found in two tumors. The fraction of mutated mtDNA in cultured lipoma cells was the same as in the lipoma in situ, indicating that the cultured cells were representative of the primary tumor. These findings indicate that the lipomas have originated with a grossly normal stem line and subsequently have developed the 6q deletion. We conclude that the lipomas represent clonal growth of adipocytes with a high content of mtDNA with the tRNA(Lys) mutation. The tRNA(Lys) mutation may be either the direct or the indirect cause of pertubation of the maturation process of the adipocytes, leading to an increased risk of lipoma formation.     

Maternally inherited cardiomyopathy and hearing loss associated with a novel mutation in the mitochondrial tRNA(Lys) gene (G8363A).

A novel G8363A mutation in the mtDNA tRNA(Lys) gene was associated, in two unrelated families, with a syndrome consisting of encephalomyopathy, sensorineural hearing loss, and hypertrophic cardiomyopathy. Muscle biopsies from the probands showed mitochondrial proliferation and partial defects of complexes I, III, and IV of the electron-transport chain. The G8363A mutation was very abundant (>95%) in muscle samples from the probands and was less copious in blood from 18 maternal relatives (mean 81.3% +/- 8.5%). Single-muscle-fiber analysis showed significantly higher levels of mutant genomes in cytochrome (c) oxidase-negative fibers than in cytochrome (c) oxidase-positive fibers. The mutation was not found in >200 individuals, including normal controls and patients with other mitochondrial encephalomyopathies, thus fulfilling accepted criteria for pathogenicity.     

Mitochondrial DNA mutation in a Chinese family with myoclonic epilepsy and ragged-red fiber disease

We analyzed the mitochondrial DNA of blood cells of 5 patients from a Chinese family with myoclonic epilepsy and ragged-red fiber disease. The results showed that in all the affected individuals there was a point mutation from A to G at the 8344th nucleotide pair, which was located in the tRNA(Lys) gene. No such a mutation was found in mtDNA of either unaffected members of that family or other healthy Chinese subjects. These findings are consistent with the recent report of Shoffner et al. (Cell 1990, 61: 931-937), and confirm that the point mutation is indeed the cause of this disease.     

Defect in modification at the anticodon wobble nucleotide of mitochondrial tRNA(Lys) with the MERRF encephalomyopathy pathogenic mutation

Hen lysozyme single-disulfide variants were constructed to characterize the structures associated with the formation of individual native disulfide bonds. Circular dichroism spectra and the effective concentration of protein thiol groups showed that the propensity for structure formation was relatively high for Cys-6–Cys-127 and Cys-30–Cys-115 disulfides. The urea concentration dependence of individual effective concentrations showed that the apparent sizes of the structures were 14–50% of the whole molecule. The intrinsic stability of each submolecular structure in a reduced form of protein, obtained by subtracting the entropic contribution of cross-linking, was highest for Cys-64–Cys-80 and lowest for Cys-76–Cys-94 disulfide bonds.     

The protective roles of phosphorylated heat shock protein 27 in human cells harboring myoclonus epilepsy with ragged-red fibers A8344G mtDNA mutation

Mitochondrial DNA (mtDNA) mutations are associated with a large number of neuromuscular diseases. Myoclonus epilepsy with ragged-red fibers (MERRF) syndrome is a mitochondrial disease inherited through the maternal lineage. The most common mutation in MERRF syndrome, the A8344G mutation of mtDNA, is associated with severe defects in mitochondrial protein synthesis, which impair the assembly and function of the respiratory chain. We have previously shown that there is a decreased level of heat shock protein 27 (HSP27) in lymphoblastoid cells derived from a MERRF patient and in cytoplasmic hybrids (cybrids) harboring the A8344G mutation of mtDNA. In the present study, we found a dramatic decrease in the level of phosphorylated HSP27 (p-HSP27) in the mutant cybrids. Even though the steady-state level of p-HSP27 was reduced in the mutant cybrids, normal phosphorylation and dephosphorylation were observed upon exposure to stress, indicating normal kinase and phosphatase activities. To explore the roles that p-HSP27 may play, transfection experiments with HSP27 mutants, in which three specific serines were replaced with alanine or aspartic acid, showed that the phosphomimicking HSP27 desensitized mutant cybrids to apoptotic stress induced by staurosporine (STS). After heat shock stress, p-HSP27 was found to enter the nucleus immediately, and with a prolonged interval of recovery, p-HSP27 returned to the cytoplasm in wild-type cybrids but not in mutant cybrids. The translocation of p-HSP27 was correlated with cell viability, as shown by the increased number of apoptotic cells after p-HSP27 returned to the cytoplasm. In summary, our results demonstrate that p-HSP27 provides significant protection when cells are exposed to different stresses in the cell model of MERRF syndrome. Therapeutic agents targeting anomalous HSP27 phosphorylation might represent a potential treatment for mitochondrial diseases.     

A novel heteroplasmic tRNA(Leu(CUN)) mtDNA point mutation associated with chronic progressive external ophthalmoplegia

We have sequenced all mitochondrial tRNA genes from a patient with chronic progressive external ophthalmoplegia (CPEO) and mitochondrial myopathy, who had no detectable large mtDNA deletions. Direct sequencing failed to detect previously reported mutations and showed a heteroplasmic mutation at nucleotide 12,276 in the tRNA(Leu(CUN)) gene, in the dihydrouridine stem, which is highly conserved through the species during evolution. RFLP analyses confirmed that 18% of muscle mtDNA harbored the mutation, while it was absent from DNA of fibroblasts and lymphocytes of the proband and in 110 patients with other encephalomyopathies. To date, besides large and single nucleotide deletions, several point mutations on mitochondrial tRNA genes have been reported in CPEO patients, but only three were in the gene coding for tRNA(Leu(CUN)).     

Identification of a new mtDNA mutation (14724G>A) associated with mitochondrial leukoencephalopathy

We report a novel 14724G>A mutation in the mitochondrial tRNA glutamic acid gene in a 4-year-old boy with myopathy and leukoencephalopathy. A muscle biopsy showed cytochrome c oxidase-negative ragged-red fibers and biochemical analysis of the respiratory chain enzymes in muscle homogenate revealed partial complex I and complex IV deficiencies. The mutation, which affects the dihydrouridine arm at a conserved site, was nearly homoplasmic in muscle and heteroplasmic in blood DNA of the proband, but it was absent in peripheral leukocytes from the asymptomatic mother, sister, and two maternal aunts, suggesting that it arose de novo. This report proposes to look for variants in the mitochondrial genome when dealing with otherwise undetermined leukodystrophies of childhood.     

A new mtDNA mutation associated with Leber hereditary optic neuroretinopathy.

A single base mutation at nucleotide position 3460 (nt 3460) in the ND1 gene in human mtDNA was found to be associated with Leber hereditary optic neuroretinopathy (LHON). The G-to-A mutation converts an alanine to a threonine at the 52d codon of the gene. The mutation also abolishes an AhaII restriction site and thus can be detected easily by RFLP analysis. The mutation was found in three independent Finnish LHON families but in none of the 60 controls. None of the families with the nt 3460 mutation in ND1 had the previously reported nt 11778 mutation in the ND4 gene. The G-to-A change at nt 3460 is the second mutation so far detected in LHON.     

Simultaneous A8344G heteroplasmy and mitochondrial DNA copy number quantification in myoclonus epilepsy and ragged-red fibers (MERRF) syndrome by a multiplex molecular beacon based real-time fluorescence PCR

The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype–phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA^Lys mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.     

A new mtDNA mutation associated with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS)

In 3 of 40 MELAS patients, a new common mutation, a T-to-C transition at nucleotide position 3271 in the mitochondrial tRNA(Leu(UUR] gene was recognized and was very near to the most common mutation site at 3243. With a simple detection method using polymerase chain reaction with a mismatch primer, none of 46 patients with other mitochondrial diseases and 50 controls had this mutation.     

A novel mutation in the mitochondrial tRNA Asn gene associated with a lethal disease

We describe a lethal mitochondrial disease in a 10-month-old child who presented with encephalomyopathy. Histochemical and electron microscopy examinations of skeletal muscle biopsy revealed abnormal mitochondria associated with a combined deficiency of complexes I and IV. After excluding mitochondrial DNA deletions and depletion, direct sequencing was used to screen for mutation in all transfer RNA (tRNA) genes. A T-to-C substitution at position 5693 in the tRNA(Asn) gene was found in blood and muscle. Microdissection of muscle biopsy and its analysis revealed the highest level of this mutation in cytochrome c oxidase (COX)-negative fibres. We suggest that this novel mutation would affect the anticodon loop structure of the tRNA(Asn) and cause a fatal mitochondrial disease.     

A novel mtDNA ND6 gene mutation associated with LHON in a Caucasian family

Leber's hereditary optic neuropathy (LHON) is a frequent cause of inherited blindness. A routine screening for common mtDNA mutations constitutes an important first in its diagnosis. However, a substantial number of LHON patients do not harbor known variants, both pointing to the genetic heterogeneity of LHON and bringing into question its genetic diagnosis. We report a familial case that exhibited typical features of LHON but lacked any of the common mutations. Genetic analysis revealed a novel pathogenic defect in the ND6 gene at 14279A that was not detected in any haplogroup-matched controls screened for it, nor has it been previously reported. This mutation causes a substantial conformational change in the secondary structure of the polypeptide matrix coil and may explain the LHON expression. Thus, it expands the spectrum of deleterious changes affecting ND6-encoding subunit and further highlights the functional significance of this gene, providing additional clues to the disease pathogenesis.     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.