Novel heteroplasmic mtDNA mutation in a family with heterogeneous clinical presentations.

The protean manifestations of a novel maternally inherited point mutation of the mitochondrial genome are reported. The proband showed isolated, spastic paraparesis. A brother, who had suffered from a multisystem progressive disorder, ultimately died of cardiomyopathy. Another brother is healthy. The proband's mother showed truncal ataxia, dysarthria, severe hearing loss, mental regression, ptosis, ophthalmoparesis, distal cyclones, and diabetes mellitus. A muscle biopsy performed in the proband failed to show the morphological abnormalities typical of mitochondrial disorders; the activities of respiratory chain complexes were normal. However, complex I and IV activities were low in the muscle homogenate of the affected mother and brother. Sequence analysis of mtDNA showed a heteroplasmic mutation of the tRNA(Ile) gene (G4284A). The mutation load was approximately 55%, 80%, and 90% in the muscle mtDNA of the proband, his mother, and his affected brother, respectively. Mutation was undetected in the healthy brother, as well as in 100 control samples. Several cybrid clones containing homoplasmic mutant mtDNA from the proband showed significant reductions of complex IV activity and maximum oxygen consumption rate, compared with homoplasmic wild-type clones derived from the same subject.     

Sporadic MERRF/MELAS overlap syndrome associated with the 3243 tRNALeu(UUR) mutation of mitochondrial DNA

We studied a patient with a mitochondrial encephalomyopathy characterized by the presence of all the cardinal features of both myoclonic epilepsy and ragged-red fibers (MERRF) and mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) syndromes. Muscle biopsy showed ragged-red fibers (RRF). Some RRF were cytochrome c oxidase (COX)-negative, while some others stained positive for COX. Muscle biochemistry revealed defects of complexes I and IV of the respiratory chain. Both muscle and blood mitochondrial DNA from the patient showed the presence of the mutation at nucleotide position 3243 in the tRNA^Leu(UUR) gene and the absence of point mutations related to MERRF syndrome. The proportions of mutant mtDNA were 70% in muscle and 30% in blood. The mutation was absent in blood from all maternal relatives, in hair follicles from the mother, and in muscle from one sister of the proband. Therefore, there was no evidence of maternal inheritance. © 1996 John Wiley & Sons, Inc.     

Rapidly progressive neurodegeneration in a case with the 7472insC mutation and the A7472C polymorphism in the mtDNA tRNA ser(UCN) gene

The authors report the clinical, neuroimaging, muscle biopsy and mtDNA findings in a patient affected by bilateral hearing loss and mental retardation since infancy, presenting at age 31 years with a rapid deterioration of mental status and ataxia leading to vegetative condition and death at the age of 32 years. Clinical and genetic studies have been also performed in the mother, affected by neurosensorial hearing loss. Muscle biopsy showed severe mitochondrial alterations in the propositus and evidence of mitochondrial alterations in his mother. Direct mtDNA sequencing in all family members revealed the known 7472insC mutation and the recently described A7472C sequence variation in the tRNA(Ser(UCN))gene. RFLP-PCR confirmed the heteroplasmic nature of the two mutations and failed to find the second transversion in 200 controls. The percentage of mutant genomes harbouring 7472insC ranged from 3 to 7% in asymptomatic family members to 70% in the proband and his mother, whereas the percentage of A7472C mutant genomes was about 90% in all maternal relatives except the proband (56%) and his sister (5%). In conclusion, this is the first report of a rapidly progressive encephalopathy in association with the 7472insC mutation in mtDNA, combined with an A>C variation at the same nucleotide with a possible suppression effect on the pathogenic mutation.     

The A to G transition at nt 3243 of the mitochondrial tRNALeu(UUR) may cause an MERRF syndrome.

OBJECTIVE--To verify the phenotype to genotype correlations of mitochondrial DNA (mtDNA) related disorders in an atypical maternally inherited encephalomyopathy. METHODS--Neuroradiological, morphological, biochemical, and molecular genetic analyses were performed on the affected members of a pedigree harbouring the heteroplasmic A to G transition at nucleotide 3243 of the mitochondrial tRNALeu(UUR), which is usually associated with the syndrome of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). RESULTS--The proband was affected by a fullblown syndrome of myoclonic epilepsy with ragged red fibres (MERRF), severe brain atrophy, and basal ganglia calcifications, without the MRI T2 hyperintense focal lesions which are pathognomonic of MELAS. Oligosymptomatic relatives were variably affected by lipomas, goitre, brain atrophy, and basal ganglia calcifications. Muscle biopsies in the proband and his mother showed a MELAS-like pattern with cytochrome c oxidase hyperreactive ragged red fibres and strongly succinate dehydrogenase reactive vessels. Quantification of the A3243G mutation disclosed 78% and 70% of mutated mtDNA in the muscle of the severely affected proband and of his oligosymptomatic mother respectively. Nucleotide sequencing of the mitochondrial tRNALeu(UUR) and tRNALys in the proband's muscle failed to show any additional nucleotide change which could account for the clinical oddity of this pedigree by modulating the expression of the primary pathogenic mutation. CONCLUSION--So far, MERRF has been associated with mutations of the mitochondrial tRNALys, and MELAS with mutations of the mitochondrial tRNALeu(UUR). Now MERRF may also be considered among the clinical syndromes associated with the A to G transition at nt 3243 of the tRNALeu(UUR).     

A novel mitochondrial ATPase 6 point mutation in familial bilateral striatal necrosis

A T-to-C transition at nucleotide (nt) 9176 in the mitochondrial adenosine triphosphatase 6 (ATPase 6) gene was detected in 2 brothers with a neurological disorder resembling Leigh syndrome. The mutation was also present in the 2 other siblings and in the mother, who were asymptomatic. In the more severely affected boy (the proband), the mutation was homoplasmic in muscle, leucocytes, and fibroblasts. In leucocytes from his affected brother, 98% of mtDNA was mutant. Heteroplasmy of varying degrees was seen in leucocytes from the mother and the 2 unaffected siblings. The mutation changes a highly conserved leucine residue near the carboxyl terminus of the mitochondrial ATPase 6 subunit to proline. It could not be detected in 168 control subjects. Studies of ATP synthesis and hydrolysis in fibroblasts from the proband were normal.     

Gene dosage effect in one family with myoclonic epilepsy and ragged-red fibers (MERRF)

Objectives - We analyzed the percentage of mitochondrial DNA (mtDNA) heteroplasmy in blood samples of 13 individuals belonging to a three family generation of myoclonic epilepsy with ragged-red fibers (MERRF) and compared the 5 affected patients and the 8 unaffected relatives. Material and methods - DNA was extracted from blood and muscle of the proband and from blood of 12 maternal relatives. A PCR restriction analysis method was used to detect the mutation. Results - The proband had the complete MERRF phenotype. The phenotype in three other individuals in the maternal lineage was consistent with the MERRF syndrome. The remaining were asymptomatic. The np 8344 mutation was observed in muscle and blood of the proband, and in blood from every one of 12 maternal relatives, ranging from 44% to 83% of mutated genomes. Symptomatic individuals had higher levels ( P < 0.001) of mutated mtDNA than asymptomatic maternal relatives. However, high proportions of mutant genomes (up to 63%) were found in asymptomatic relatives. Conclusions - Although there seems to be a gene dosage effect in MERRF, we found no absolute relationship between the relative proportion of mutant genomes in blood and clinical severity. Factors other than gene dosage in blood may account for the differences in clinical phenotype.     

A novel heteroplasmic tRNA(Ser(UCN)) mtDNA point mutation associated with progressive external ophthalmoplegia and hearing loss

We sequenced all mitochondrial tRNA genes from a patient with sporadic external ophthalmoplegia (PEO) and 5% COX-negative fibers in muscle biopsy, who had no detectable large mtDNA deletions. Direct sequencing showed a heteroplasmic mutation at nucleotide 7506 in the dihydrouridine stem of the tRNA(Ser(UCN)) gene. RFLP analysis confirmed that 30% of muscle and 20% of urinary epithelium mtDNA harbored the mutation, which was absent in other tissues of the proband as well as in mtDNA of his mother and 100 patients with various encephalomyopathies. Several point mutations on mitochondrial tRNA genes have been reported in PEO patients without large-scale rearrangements of mtDNA but no point mutations have hitherto been found in the gene coding for tRNA(Ser(UCN)).     

MELAS syndrome: correlation between clinical features and molecular genetic analysis

The clinical manifestations and mitochondrial DNA (mtDNA) mutations in a Taiwanese family with a female proband exhibiting mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes syndrome are reported. Clinically, the proband had a stroke-like episode with right hemiparesis, hemianopsia and mental dysfunction as well as short stature, hearing impairments, and elevated lactate levels. Brain magnetic resonance images showed multiple increased signal intensities over the left frontal, parietal and temporal areas. There were no ragged-red fibers, but paracrystalline inclusion bodies were shown in the muscle biopsies under electron microscopic examination. A deficiency of NADH-CoQ reductase was also found in biochemical studies of the muscles. The family survey revealed no abnormal findings except for headache and episodic vomiting in her mother. The molecular analysis of mtDNA disclosed a mutation from A to G at the nucleotide pair 3243 of the mitochondrial transfer RNA^Leu gene in the blood, hair follicles and/or muscle of the maternal relatives. A characteristic finding of the MELAS family is variation of percentage of mutated mtDNA in various tissues and individuals. However, a higher proportion of mutated mtDNA was noted in the proband than that in the asymptomatic or oligosymptomatic family members. From the data, the variable clinical phenotypes in this MELAS family can be explained at least partly, by the different proportions of mutant mtDNA in the target tissues of the profound and maternal relatives.     

A novel mutation (8342G-->A) in the mitochondrial tRNA(Lys) gene associated with progressive external ophthalmoplegia and myoclonus

We describe a patient who suffered from impaired ocular motility from age 10 years and at 16 years developed ptosis, proximal weakness and progressive fatigability. At 35 years she developed massive myoclonic jerks, and head and distal tremor. A muscle biopsy showed a high percentage of cytochrome c oxidase negative fibers but no ragged-red fibers. A novel heteroplasmic mutation (8342G-->A) was found in the mitochondrial transfer RNA(Lys) gene by single-strand conformation polymorphism screening, followed by sequence and restriction fragment length polymorphism analysis. Approximately 80% of muscle mitochondrial DNA (mtDNA) harbored the mutation, while the mutation was absent in lymphocyte DNA of the proband, as well as of her mother, daughter and a maternal aunt. However, the pathogenicity of the mutation was confirmed by restriction fragment length polymorphism analysis of single muscle fibers, which revealed a significantly greater level of mutant mtDNA in cytochrome c oxidase negative over cytochrome c oxidase positive fibers.     

Tissue mosaicism in the skeletal muscle and sural nerve biopsies in the MELAS syndrome

We describe a clinically full-blown MELAS patient, who had an A3243G point mutation of mitochondrial DNA (mtDNA) in muscle and blood cells, and his family members. From the proband two muscle biopsies from the vastus lateralis muscle were analysed; one had typical ragged red fibers and focal cytochrome c oxidase deficiency and the other was completely normal. He also had a peripheral neuropathy confirmed by nerve conduction velocity and sural nerve biopsy studies. Axonal degeneration, relative loss of large myelinated fibers and paracrystalline inclusion bodies in the Schwann cells were noted. Intriguingly, the A3243G mutation of mtDNA was not found in the sural nerve biopsy. Therefore, we conclude that tissue mosaicism is present in the muscle fibers and that the mtDNA mutation may not be detected in the nerve involved as proved by pathology. We also suggest that the involvement of specific tissues in patients with mitochondrial diseases should be further determined by single fiber mtDNA analysis.     

A novel mutation in the mitochondrial tRNAVal gene associated with a complex neurological presentation

We describe a patient who presented with progressive ataxia, sezures, mental deterioration, mild myopathy, and hearing loss. A novel heteroplasmic G-to-A transition was found, affecting the acceptor stem of the mitochondrial (mt) tRNA^Val gene. Mutant mtDNA was 67% of total mtDNA in the muscle of the proband and was also present at low levels in the muscle of his healthy mother. It was absent in all of the numerous control DNA samples that were tested. Analysis of single muscle fibers revealed a significantly greater level of mutant mtDNA in cytochrome c oxidase-negative fibers. Mutations of mtDNA may be responsible of neurological syndromes that, like the case reported here, are clinically puzzling, and lack typical “mitochondrial” clues, such as elevated levels of blood lactate, overt defects of the respiratory complexes, and clinically documented maternal inheritance.     

The mitochondrial DNA A3243G mutation in Portugal: clinical and molecular studies in 5 families.

Out of 90 Portuguese patients with mitochondrial cytopathy, six harbored the A3243G mutation in the mtDNA tRNA(Leu(UUR)) gene ('MELAS mutation'). They had heterogeneous clinical features, including myopathy with stroke-like episodes, progressive external ophthalmoparesis, diabetes mellitus, and subacute encephalopathy. Histochemical and biochemical analyses of muscle biopsies showed abundant ragged-red fibers reacting positively with the cytochrome oxidase stain, and decreased respiratory chain enzyme activities. On average, the proportion of mutated mtDNA was 67% (20-88%) in tissues from patients and 21% (0-49%) in blood from 20 maternal relatives. The proportion of mutated mitochondrial genomes in muscle did not correlate with clinical presentation or duration of disease. This study, the first in Portuguese patients, confirms the frequent occurrence of the A3243G mutation in patients with mitochondrial diseases, and emphasises the usefulness of genetic testing in reaching a correct diagnosis.     

A novel mitochondrial tRNA phenylalanine mutation presenting with acute rhabdomyolysis

We describe a patient who presented with acute rhabdomyolysis and had 68% cytochrome c oxidase (COX)-deficient fibers in skeletal muscle. Further investigations confirmed a respiratory chain defect that was associated with a novel heteroplasmic point mutation in the phenylalanine tRNA gene of the mitochondrial genome (mtDNA). Analysis of single muscle fibers revealed a significantly greater level of mutant mtDNA in COX-negative fibers. This is the first case of a mitochondrial tRNA gene point mutation presenting with acute rhabdomyolysis and recurrent myoglobinuria.     

Clinical and genetic features in two families with MELAS and the T3271C mutation in mitochondrial DNA

The majority of patients with MELAS (mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes) have the A3243G point mutation. The much rarer T3271C mutation has been reported predominantly in Japanese subjects. Our objective was to better define the clinical phenotype and mutation load in patients with MELAS and the T3271C mutation in mitochondrial DNA. We present clinical and molecular genetic data in two pedigrees with the T3271C mutation. The age at onset was 8 years in one proband and 14 years in the other. Both patients had migrainelike headache, seizures, and strokelike episodes. Mutation loads were quantified in multiple tissues from the patients and from family members by polymerase chain reaction-restriction fragment length polymorphism analysis. The symptoms in both probands were typical of MELAS, and, contrary to previous reports, onset was early. Hearing loss was less common than in typical MELAS, and ragged red fibers were absent. The proportion of mutant genomes was consistently and markedly greater in DNA from urinary sediment than from blood. In the mother of one proband, mutant genomes were detected only in DNA from hair follicles and cheek mucosa The phenotype of patients with the T3271C mutation might not be as distinct as that of the A3243G mutation, as previously described. Our data also suggest that urine is a better source of DNA than blood for diagnosis and that multiple tissues should be studied in maternal relatives, especially when the mutation cannot be detected in blood.     

Phenotypes and mitochondrial DNA substitutions in families with A3243G mutation

OBJECTIVE: To clarify the relationship between mitochondrial DNA (mtDNA) sequence variations and phenotypes in patients with A3243G mutation. MATERIALS AND METHODS: We studied whole mtDNA sequences in two families with A3243G mutation and characteristic clinical features. Two brothers in Family 1 had shown thiamine deficiency and mitochondrial myopathy without central nervous system involvement. In Family 2, a 16-year-old woman showed the symptoms of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Her mother had had diabetes mellitus and died at the age of 42. The proportion of A3243G mtDNA in blood was 87 and 89% in the patients of Family 1, and 25% in the patient and less than 5% in the mother of Family 2. RESULTS: The mtDNA analysis revealed the following homoplasmic substitutions: T1520C and C12153T found only in Family 1, and A15954G found only in Family 2. These substitutions were not detected in seven other MELAS patients or in 50 controls. CONCLUSION: These substitutions might be specific to these families and could be one of the factors that modulate their clinical features together with the A3243G mutation.     

Single muscle fiber analysis of myoclonus epilepsy with ragged-red fibers

We examined two muscle biopsy specimens from a proband and her mother with myoclonus epilepsy with ragged-red fibers (MERRF), both obtained at an interval of about 10 years, using histochemistry, in situ hybridization, and single-fiber polymerase chain reaction. Total (wild-type and mutant) mitochondrial DNAs (mtDNAs) were greatly increased in ragged-red fibers (RRF) over non-RRF in all muscle specimens analyzed. The proportion of mutant mtDNA was also significantly higher in RRF than in non-RRF. By comparing the first and second muscle biopsied specimens in each patient, we found that while the proportion of RRF, cytochrome c oxidase deficient fibers, and mutant DNA in muscle changed over a 10-year period, the proportion of wild-type and mutant mtDNAs in RRF and in non-RRF was similar between the two specimens. These results suggest that the ratio of wild-type to mutant mtDNAs in RRF and non-RRF in MERRF is at a steady state level in each muscle fiber, without replicative advantage of mutant mtDNA. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:490–497, 1998.     

MELAS- and kearns-sayre-type with myopathy and autoimmune polyendocrinopahy

A 35-year-old woman with features of Kearns-Sayre syndrome consisting of progressive ptosis, ophthalmoparesis, mitochondrial myopathy, and pigmentary retinopathy also had autoimmune polyglandular syndrome type I1 (Addison's disease, autoimmune insulin-dependent diabetes meuitus, Hashimoto's thyroiditis, and primary ovarian failure). There was no history of similarly affected relatives. Analysis of muscle mitochondrial DNA (mtDNA) revealed a 2,532-bp deletion of the type seen in Kearns-Sayre syndrome as well as a heteroplasmic A3243G mutation in the tRNA-Leu(UUR) gene of the type seen in mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes (MELAS). The patient's blood and her mother's blood harbored the A3243G mutation but not the deletion, and the maternal grandmother's blood had neither mutation. In muscle, the species of mtDNA harboring the deletion was exclusively associated with the species harboring the A3243G mutation, suggesting that the point mutation predisposed to the large-scale deletion. The mtDNA species with both mutations accounted for 88% of total muscle mtDNA. Other and as yet unrecognized point mutations in mtDNA might also be associated with, and possible causally related to, largescale mtDNA deletions.     

"MELAS" (A3243G) mutation of mitochondrial DNA: a study of the relationships between the clinical phenotype in 19 patients and morphological and molecular data

Nineteen patients were found to harbor the mitochondrial DNA A3243G mutation associated with MELAS syndrome (Mitochondrial myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes). Eight of them had presented with stroke-like episodes and therefore had a clinical diagnosis of MELAS syndrome. The other 11 patients had no strokes and presented with generally less severe multisystemic disease. In the two groups, we compared muscle morphology, biochemical activities of muscle respiratory chain, and genetic characteristics: proportion and tissue distribution of the mutation, sequence of the 22 transfer RNA genes of the mitochondrial DNA. The proportion of mutant mtDNA in muscle was always greater than in blood. The number of patients in the two groups was too low to reach significant values. However, the patients with a MELAS syndrome presented with more severe respiratory chain abnormalities and with a proportion of the A3243G mutation that was both higher and more uniformly distributed among tissues. For symptoms others than stroke-like episodes, we did not observe any correlation with the level of mutant mtDNA in muscle. The analysis of the 22 tRNA sequences did not show differences between the two groups, and no co-inherited modifying tRNA genes could explain the variability of severity in our patients.     

MELAS syndrome associated with a tandem duplication in the D-loop of mitochondrial DNA

We describe a family with two cases of adult-onset mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Interestingly, the proband also had non-insulin dependent diabetes mellitus and hyperthyroidism. Endocrinological studies demonstrated a high titer of TSH receptor antibody in the proband and elevated levels in her maternal relatives. Analysis of mitochondrial DNA (mtDNA) showed an A-to-G transition at nucleotide position 3243 in the tRNA^Leu(UUR) gene (A3243G) in the three generations of the family. Furthermore, a previously described ~ 260 bp tandem duplication in the D-loop region of mtDNA was also found in the proband and her maternal relatives. To our knowledge, such kind of duplication has never before been reported in the MELAS syndrome. The proportions of mtDNA with the ~260 bp tandem duplication and A3243G point mutation were 12.5% and 82% in the muscle, respectively, and 1.6% and 35% in the blood cells, respectively, of the proband. We conclude that the hyperthyroidism in this MELAS patient may be related to the tandem duplication in the D-loop of mtDNA. This study further substantiates the importance of searching for additional genetic mutations in mitochondrial encephalomyopathic patients with new clinical phenotypes.