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.     

Mitochondrial DNA mutations in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS)

The total sequences of mitochondrial DNA were determined in two patients with juvenile-onset mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) due to Complex I deficiency. Patients 1 and 2 had three and two unique point mutations, respectively, causing replacement of phylogenically conserved amino acids. A transition from G to A was found at nucleotide position 5601 in the alanine tRNA gene of Patient 2, and a transition from A to G was found at 3243 in the leucine (UUR) tRNA gene of both patients. The latter mutation located at the phylogenically conserved 5' end of the dihydrouridine loop of the tRNA molecule, and was present in two patients with adult-onset MELAS and absent in controls. These results indicate that a mass of mtDNA mutations including the A-to-G transition in the tRNA(Leu) gene is a genetic cause of MELAS.     

Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes

The mitochondrial tRNA(Leu)(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAs(Leu)(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAs(Leu)(UUR) versus uncharged tRNAs(Leu)(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAs(Leu)(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.     

New mitochondrial tRNA HIS mutation in a family with lactic acidosis and stroke-like episodes (MELAS)

We report a new mutation in m.12146 A > G in the mt-tRNA^His in a family with a remarkable clinical history having different degrees of lactic acidosis and stroke-like episodes. Biochemical measurements of a muscle biopsy established an isolated complex IV deficiency, while similar analysis of fibroblasts showed a combined complex I,III and IV deficiency. Transmitochondrial cybrid analysis proved that this tRNA^His mutation causes the enzymatic deficiency. This family illustrates the complexity of the clinical, biochemical and genetic characteristics of a novel mtDNA encoded disorder, as well as the challenge to prove its pathogenicity.     

Respiration-deficient cells are caused by a single point mutation in the mitochondrial tRNA-Leu (UUR) gene in mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS).

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) is a major subgroup of heterogeneous mitochondrial diseases. For identifying a mutation in the mitochondrial gene, we isolated, from the same muscle tissue from a patient with MELAS, cell lines with distinctly different phenotypes: one was respiration-deficient, and the other was apparently normal. Compared with the normal cells, only one A-to-G nucleotide transition at nucleotide 3243 in the tRNA-Leu (UUR) gene was found in whole mtDNA of the respiration-deficient cells. This mutation was also found in eight patients, from unrelated families, who had MELAS in a heteroplasmic manner but was not found in control individuals. Therefore, the single point mutation causes the functional abnormality in the respiratory chain of mitochondria.     

Mutation in the mitochondrial tRNA(Val) causes mitochondrial encephalopathy, lactic acidosis and stroke-like episodes

An m.1630A>G mutation in the mitochondrial tRNA(Val) (MTTV) was identified in a patient with hearing impairment, short stature and new onset of stroke. This mutation has previously been identified in a patient with the mitochondrial neurogastrointestinal encephalopathy syndrome (MNGIE). The mother of the proband also had high levels of the m.1630A>G allele present in blood and other tissues, without symptoms. To confirm the pathogenicity of this mutation, we created cybrid cell lines with various mutation loads. The m.1630A>G mutation impairs oxygen consumption, affects the stability of the MTTV and reduces the levels of subunits of the electron transport chain.     

An analysis of the clinical and imaging features of mitochondrial encephalopathy,lactic acidosis,and stroke-like episodes (MELAS)

Abstract

Objective: To investigate the clinical features and imaging characteristics of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS).

Methods: Seventeen patients with MELAS diagnosed in the Affiliated Hospital of Xuzhou Medical University from July 2014 to August 2018 were enrolled in this study and their clinical manifestations, imaging and histopathological features were retrospectively analysed. We also discussed and summarised the related literature.

Results: All of the 12 patients had seizures; stroke-like episodes in 12 cases; audio-visual impairment in 12 cases; headache in six cases; dysplasia in four cases; mental retardation in three cases; ataxia in two cases. On cranial magnetic resonance (MR) scans, the most common manifestations were in temporal–occipital–parietal lobe, cortical or subcortical areas as well as frontal lobe, thalamus, and basal ganglia showing long or equal T1 signals, long T2 signals, and hyperintense or iso-intense diffusion-weighted imaging (DWI) signals accompanied by ventricular enlargement and brain atrophy. MR spectroscopy showed that lactic acid peaks could be found in lesion sites, normal brain tissues, and cerebrospinal fluid. Muscle biopsy and genetic testing are the gold standard for diagnosing MELAS, muscle biopsy revealed COX-negative muscle fibres and SDH-stained red ragged fibres (RRF) under the sarcolemma. Mutations of mtDNA A3243G locus were common on gene testing. Improvement of mitochondrial function was observed after symptomatic and supportive treatment.

Conclusion: MELAS should be considered for patients with epileptic seizures, headache, stroke-like episodes, extraocular palsy, cognitive decline and other clinical manifestations with the lesion located in the temporal–occipital–parietal lobe regardless of the distribution of blood vessels, and further examinations including muscle biopsy and gene testing should be performed to confirm the diagnosis.     

Overexpressed mitochondrial leucyl-tRNA synthetase suppresses the A3243G mutation in the mitochondrial tRNA(Leu(UUR)) gene

The A3243G mutation in the human mitochondrial tRNA^Leu(UUR) gene causes a number of human diseases. This mutation reduces the level and fraction of aminoacylated tRNA^Leu(UUR) and eliminates nucleotide modification at the wobble position of the anticodon. These deficiencies are associated with mitochondrial translation defects that result in decreased levels of mitochondrial translation products and respiratory chain enzyme activities. We have suppressed the respiratory chain defects in A3243G mutant cells by overexpressing human mitochondrial leucyl-tRNA synthetase. The rates of oxygen consumption in suppressed cells were directly proportional to the levels of leucyl-tRNA synthetase. Fifteenfold higher levels of leucyl-tRNA synthetase resulted in wild-type respiratory chain function. The suppressed cells had increased steady-state levels of tRNA^Leu(UUR) and up to threefold higher steady-state levels of mitochondrial translation products, but did not have rates of protein synthesis above those in parental mutant cells. These data suggest that suppression of the A3243G mutation occurred by increasing protein stability. This suppression of a tRNA gene mutation by increasing the steady-state levels of its cognate aminoacyl-tRNA synthetase is a model for potential therapies for human pathogenic tRNA mutations.     

A new disease-related mutation for mitochondrial encephalopathy lactic acidosis and strokelike episodes (MELAS) syndrome affects the ND4 subunit of the respiratory complex I.

The molecular lesions in two patients exhibiting classical clinical manifestations of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) syndrome have been investigated. A recently reported disease-related A----G base substitution at nt 3243 of the mtDNA, in the DHU loop of tRNA(Leu), was detected by restriction-enzyme analysis of the relevant PCR-amplified segment of the mtDNA of one patient but was not observed, by either restriction-enzyme analysis or nucleotide sequencing, in the other. To define the molecular lesion in the patient who does not have the A----G base substitution at nt 3243, the total mitochondrial genome of the patient has been sequenced. An A----G base substitution at nt 11084, leading to a Thr-to-Ala amino acid replacement in the ND4 subunit of the respiratory complex I, is suggested to be a disease-related mutation.     

The diabetes-associated 3243 mutation in the mitochondrial tRNA(Leu(UUR)) gene causes severe mitochondrial dysfunction without a strong decrease in protein synthesis rate.

Cells harboring patient-derived mitochondria with an A-to-G transition at nucleotide position 3243 of their mitochondrial DNA display severe loss of respiration when compared with cells containing the wild-type adenine but otherwise identical mitochondrial DNA sequence. The amount and degree of leucylation of tRNA(Leu(UUR)) were both found to be highly reduced in mutant cells. Despite the low level of leucyl-tRNA(Leu(UUR)), the rate of mitochondrial translation was not seriously affected by this mutation. Therefore, decrease of mitochondrial protein synthesis as such does not appear to be a necessary prerequisite for loss of respiration. Rather, the mitochondrially encoded proteins seem subject to elevated degradation, leading to a severe reduction in their steady state levels. Our results favor a scheme in which the 3243 mutation causes loss of respiration through accelerated protein degradation, leading to a disequilibrium between the levels of mitochondrial and nuclear encoded respiratory chain subunits and thereby a reduction of functional respiratory chain complexes. The possible mechanisms underlying the pathogenesis of mitochondrial diabetes is discussed.     

The pathogenic U3271C human mitochondrial tRNA(Leu(UUR)) mutation disrupts a fragile anticodon stem

The U3271C mutation affecting the human mitochondrial transfer RNA(Leu(UUR)) (hs mt tRNA) is correlated with diabetes and mitochondrial encephalopathies. We have explored the relationship between the structural effects of this mutation and its impact on function using chemical probing experiments and in vitro aminoacylation assays to investigate a series of tRNA constructs. Chemical probing experiments indicate that the U3271C substitution, which replaces an AU pair with a CA mispair, significantly destabilizes the anticodon stem. The introduction of a compensatory A3261G mutation reintroduces base pairing at this site and restores the structure of this domain. In fact, the anticodon stem of the A3261G/U3271C mutant appears more structured than wild-type (WT) hs mt tRNA(Leu(UUR)), indicating that the entirely AU stem of the native tRNA is intrinsically weak. The results of the chemical probing experiments are mirrored in the aminoacylation activities of the mutants. The U3271C substitution decreases aminoacylation reactivity relative to the WT tRNA due to an increase in K(m) for the pathogenic mutant. The binding defect is a direct result of the structural disruption caused by the pathogenic mutation, as the introduction of the stabilizing compensatory mutation restores aminoacylation activity. Other examples of functional defects associated with the disruption of weak domains in hs mt tRNAs have been reported, indicating that the effects of pathogenic mutations may be amplified by the fragile structures that are characteristic of this class of tRNAs.     

Maternally transmitted diabetes mellitus associated with the mitochondrial tRNA(Leu(UUR)) A3243G mutation in a four-generation Han Chinese family

We report here the characterization of a four-generation Han Chinese family with maternally transmitted diabetes mellitus. Six (two males/four females) of eight matrilineal relatives in this family exhibited diabetes. The age of onset in diabetes varies from 15 years to 33 years, with an average of 26 years. Two of affected matrilineal relatives also exhibited hearing impairment. Molecular analysis of mitochondrial DNA (mtDNA) showed the presence of heteroplasmic tRNA(Lue(UUR)) A3243G mutation, ranging from 35% to 58% of mutations in blood cells of matrilineal relatives. The levels of heteroplasmic A3243G mutation seem to be correlated with the severity and age-at-onset of diabetes in this family. Sequence analysis of the complete mitochondrial genome in this pedigree revealed the presence of the A3243G mutation and 38 other variants belonging to the Eastern Asian haplogroup M7C. However, none of other mtDNA variants are evolutionarily conserved and implicated to have significantly functional consequence. Thus, the A3243G mutation is the sole pathogenic mtDNA mutation associated with diabetes in this Chinese family.     

Mitochondrial tRNA gene mutations in patients having mitochondrial disease with lactic acidosis

Lactic acidosis has been associated with a variety of clinical conditions and can be due to mutation in nuclear or mitochondrial genes. We performed mutations screening of all mitochondrial tRNA genes in 44 patients who referred as hyperlactic acidosis. Patients showed heterogeneous phenotypes including Leigh disease in four, MELAS in six, unclassified mitochondrial myopathy in 10, cardiomyopathy in five, MERRF in one, pure lactic acidosis in six, and others in 12 including facio-scaplo-femoral muscular dystrophy (FSFD), familial cerebellar ataxia, recurrent Reye syndrome, cerebral palsy with mental retardation. We measured enzymatic activities of pyruvate dehydrogenase complex, and respiratory chain enzymes. All mitochondrial tRNA genes and known mutation of ATPase 6 were studied by single strand conformation polymorphism (SSCP), automated DNA sequence and PCR-RFLP methods. We have found one patient with PDHC deficiency and six patients with Complex I+IV deficiency, though the most of the patients showed subnormal to deficient state of respiratory chain enzyme activities. We have identified one of the nucleotide changes in 29 patients. Single nucleotide changes in mitochondrial tRNA genes are found in 27 patients and one in ATPase 6 gene in two patients. One of four pathogenic point mutations (A3243G, C3303T, A8348G, and T8993G) was identified in 12 patients who showed the phenotype of Leigh syndrome, MELAS, cardimyopathy and cerebral palsy with epilepsy. Seventeen patients have one of the normal polymorphisms in the mitochondrial tRNA gene reported before. SSCP and PCR-RFLP could detect the heteroplasmic condition when the percentage of mutant up to 5, however, it cannot be observed by direct sequencing method. It is important to screen the mtDNA mutation not only by direct sequence but also by PCR-RFLP and the other sensitive methods to detect the heroplasmy when lactic acidosis has been documented in the patients who are not fulfilled the criteria of mitochondrial disorders.     

A whole mitochondrial genome screening in a MELAS patient: a novel mitochondrial tRNA(Val) mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA(Val). This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.     

A whole mitochondrial genome screening in a MELAS patient: A novel mitochondrial tRNA mutation

Mitochondrial encephalopathy, lactic acidosis and strokelike episodes (MELAS) syndrome is a mitochondrial disorder characterized by a wide variety of clinical presentations and a multisystemic organ involvement. In this study, we report a Tunisian girl with clinical features of MELAS syndrome who was negative for the common m.3243A>G mutation, but also for the reported mitochondrial DNA (mtDNA) mutations and deletions. Screening of the entire mtDNA genome showed several known mitochondrial variants besides to a novel transition m.1640A>G affecting a wobble adenine in the anticodon stem region of the tRNA^Val. This nucleotide was conserved and it was absent in 150 controls suggesting its pathogenicity. In addition, no mutations were found in the nuclear polymerase gamma-1 gene (POLG1). These results suggest further investigation nuclear genes encoding proteins responsible for stability and structural components of the mtDNA or to the oxidative phosphorylation machinery to explain the phenotypic variability in the studied family.