Inheritance and expression of mitochondrial DNA point mutations.

An important feature of the mitochondrial genom is the occurrence of heteroplasmy and the possibility for transmission to the offspring of various proportions of wild-type and mutated mtDNA. We have investigated the proportion of the tRNALys A8344G mutation, the tRNALeu(UUR) A3243G mutation, and the ATPase 6 T8993G mutation in patients with MERRF, MELAS, and Leigh's syndrome and their maternal relatives. The level of mutated mtDNA in the offspring of carriers of the tRNALys mutation is correlated to the level in lymphocytes in the mother and seems to be transmitted by an essentially random mechanism where only a few mtDNA copies are founders of the mitochondrial genom in the offspring and the probability that the mutation is not transmitted to the offspring is high when the mothers carriers predominantly wild-type mtDNA. However, we found age-related differences in the distribution of mutated mtDNA in carriers of the tRNALys and tRNALeu mutations, which have to be considered before levels of mutated mtDNA are used for prediction of prognosis and transmission of a disorder.

Automatic sequencing of mitochondrial tRNA genes in patients with mitochondrial encephalomyopathy.

We have investigated nine children with infantile onset of mitochondrial myopathy and two adults with myoclonus epilepsy and ragged-red fibers (MERRF) and chronic progressive external ophthalmoplegia (CPEO), respectively. These patients lacked any of the previously known pathogenic tRNA mutations. Southern blot analysis of muscle mtDNA revealed no deletions. The tRNA genes of muscle mtDNA were sequenced. Restriction enzyme analysis of PCR fragments was performed to verify the presence of the mutations identified by automatic sequencing. Several tRNA mutations were found, but they were all homoplasmic. Furthermore, the mutations were either present in controls or did not change nucleotides conserved between species. This strongly suggests that none of the tRNA mutations identified in the 11 patients with mitochondrial encephalomyopathy was pathogenic. It can thus be concluded that mitochondrial tRNA mutations and mtDNA deletions probably are an infrequent cause of mitochondrial disorders in infants. Patients with MERRF and CPEO may lack both pathogenic point mutations of tRNA genes and deletions of mtDNA.

EEG findings in children and adolescents with mitochondrial encephalomyopathies: a study of 25 cases.

EEG was studied in 25 children and adolescents with mitochondrial encephalomyopathies, defined on the basis of clinical, biochemical and morphological criteria. Twenty cases conformed to well-known mitochondrial syndromes: Alpers syndrome [6], Leigh syndrome [2], MERRF (myoclonus epilepsy and ragged red fibers) syndrome [3], MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) syndrome [5] and Kearns-Sayre syndrome [4]. Many patients were followed for several years with repeated EEG. In all, 112 EEG records were included in the study. A common feature of all the mitochondrial encephalomyopathic syndromes was slowing of the alpha rhythm. Epileptic discharges were seen in most syndromes. In spite of the small number of cases in each group, in Alpers, MERRF and MELAS syndromes we found sequential EEG patterns which seemed to be typical of the respective syndromes. In contrast, in Kearns-Sayre syndrome, a slow background rhythm was the only consistent finding. We conclude that EEG, especially repeated recordings, may be of help in the diagnostic evaluation of mitochondrial encephalomyopathies.

Early-onset myopathy with tubular aggregates.

We report a 14-year-old girl with early onset of slowly progressive muscular weakness and atrophy. There was no family history of neuromuscular disease. A persistent increase of serum creatine kinase was found. Muscle biopsy specimens showed type 1 fiber predominance and tubular aggregates in almost every fiber. The clinical findings and pathology suggest that the disease represents one variant in a group of rare myopathies with different patterns of inheritance, characterized by slowly progressive muscle weakness and tubular aggregates.

Mitochondrial myopathy and cardiomyopathy in siblings.

Two siblings with infantile lactic acidosis and mitochondrial myopathy are described. The first child, a girl, died at 5 months of age from severe lactic acidosis after about 3 weeks of progressive muscular hypotonia. The younger brother had congenital lactic acidosis but no other symptoms until 6 months of age when progressive muscle weakness appeared. Treatment with dichloroacetate lowered the serum lactic acid level but did not affect his clinical condition. At 13 months of age, cardiomyopathy was diagnosed and he died at the age of 29 months of circulatory failure. Both children had mitochondrial myopathy. Postmortem examination of the boy revealed marked morphologic changes of the mitochondria in both skeletal muscle and the myocardium; biochemical investigation of skeletal muscle mitochondria demonstrated deficiencies in both complex I (NADH ferricyanide reductase) and complex IV (cytochrome c oxidase). The disease in these siblings differs in several respects from previously reported patients with mitochondrial myopathy and cytochrome c oxidase deficiency.

Mitochondrial encephalomyopathies in childhood. I. Biochemical and morphologic investigations.

During a 4-year period (1984 to 1988), 50 children referred with manifestations of central nervous system or neuromuscular disease combined with hyperlactatemia were subjected to investigations that aimed to identify and characterize children with mitochondrial disorders. Biochemical and morphologic investigations of quadriceps muscle biopsy tissue were done, including oximetric and spectrophotometric analysis of the respiratory chain function, enzyme histochemistry, electron microscopy, and analysis of mitochondrial DNA. A diagnosis of mitochondrial disease was based on the presence of at least two of five criteria: (1) abnormal results of oximetry, (2) abnormal results of spectrophotometry, (3) enzyme histochemical evidence of cytochrome x oxidase deficiency, (4) deletions or point mutations of mitochondrial DNA, and (5) abundant ultrastructurally abnormal mitochondria. With the combined biochemical and morphologic investigation, 20 of the children were found to have mitochondrial disorders. In an additional 10 children a mitochondrial disorder was neither excluded nor verified. Mitochondrial disorders are thus an important cause of central nervous system and neuromuscular disease in children with hyperlactatemia.

Mitochondrial encephalomyopathies in childhood. II. Clinical manifestations and syndromes.

During a 4-year period 1984 to 1988, 20 children referred with manifestations of central nervous system or neuromuscular disease combined with hyperlactatemia were found to have a mitochondrial disease. Each diagnosis was based on the results of thorough biochemical and morphologic investigations. The patients were separated into one series with mainly encephalopathy (n = 14) and another with mainly myopathy (n = 6). The patients with encephalopathy had the following syndromes: Kearns-Sayre (n = 2), MERRF (myoclonus epilepsy and ragged red fibers; n = 2), MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes; n = 3), Alpers (n = 3), Leigh (n = 1), and other variants (n = 3). In patients with myopathy, three had hypertrophic nonobstructive cardiomyopathy. Ultrastructural abnormalities of mitochondria were the most common morphologic changes in the muscle biopsies. Complex I deficiency was most common in the patients with encephalopathy. All of the patients with myopathy had complex IV deficiency. Mutations of mitochondrial DNA were found in six patients with encephalopathy. We conclude that identification of defects at the DNA level and determination of the phenotypic expression with clinical, morphologic, and biochemical methods are fundamental for future rational classification of mitochondrial disorders.

Pathogenetic aspects of the A8344G mutation of mitochondrial DNA associated with MERRF syndrome and multiple symmetric lipomas.

Myoclonus epilepsy and ragged-red fibers syndrome (MERRF) is caused by a heteroplasmic mutation at nucleotide 8344 (A8344G) of the tRNA(Lys) gene of mitochondrial DNA (mtDNA). This mutation impairs mitochondrial protein synthesis and causes a respiratory chain dysfunction. The risk for transmission of the A8344G mutation from mother to child is dependent on the levels of mutated mtDNA in the mother and above a threshold level of 35-40% the mutation is transmitted to all children. The progression of symptoms in MERRF can be explained by a gene dosage effect with accumulation over time of mutated mtDNA. High levels of mutated mtDNA, ultrastructurally abnormal mitochondria, and a clonal deletion on chromosome 6 are found in lipomas associated with MERRF. These findings indicate that there is a respiratory chain dysfunction in the lipomas and that lipomas may be a manifestation of the A8344G mutation.

Atypical presentation of multisystem disorders in two girls with mitochondrial DNA deletions.

UNLABELLED: We describe two girls with atypical presentations of multisystem disorders due to deletions in mitochondrial DNA (mtDNA). One presented with painful carpopedal spasms due to hypoparathyroidism at the age of 4 years. The disease was rapidly progressive with development of truncal and limb ataxia, spastic paraparesis, muscle weakness and wasting, pigmentary retinal degeneration and sensorineural hearing loss. She had short stature and vitiligo patches, hirsutism, anaemia, diabetes mellitus and exocrine pancreatic dysfunction. The other girl presented at the age of 6 years with polydipsia, polyuria and fatigue due to renal tubular dysfunction. The disease was insidiously progressive with poor growth and development of sensorineural hearing loss, muscle weakness and truncal and limb ataxia. Morphological, enzyme histochemical and biochemical investigations indicated mitochondrial dysfunction of skeletal muscle, liver and kidney in one patient and of skeletal muscle and liver in the other. Both patients had large proportions of mtDNA molecules with deletion in liver, kidney, skeletal muscle and blood cells. CONCLUSION: It may be concluded that symptoms from several different organs may be the first manifestation of a mtDNA deletion disorder.

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.

De novo mutation in the mitochondrial ATP synthase subunit 6 gene (T8993G) with rapid segregation resulting in Leigh syndrome in the offspring.

The mutation in the mitochondrial ATP synthase subunit 6 gene (ATP6 T8993G) was identified in a male infant who died at age 15 months of Leigh syndrome. He had 94% mutated mitochondrial DNA (mtDNA) in muscle and 92% in lymphocytes. His mother was healthy but had 37% mutated mtDNA in muscle and 38% in lymphocytes. The proband's brother, who was also healthy, had 44% mutated mtDNA in lymphocytes. No mutated mtDNA was detected in muscle and lymphocytes from the maternal grandmother of the proband or in lymphocytes from 15 other maternal relatives, showing that the first carrier of the ATP6 T8993G mutation in this family was the mother of the proband. This study shows that this point mutation may occur at substantial levels in a carrier of a de novo mutation and rapid segregation with high levels of mutated mtDNA causing neurodegenerative disease may occur in the second generation.

Lack of transmission of deleted mtDNA from a woman with Kearns-Sayre syndrome to her child.

We have investigated the daughter of a woman with Kearns-Sayre syndrome. The woman had a high percentage of deleted mtDNA in muscle, but no deleted mtDNA was detected in fibroblasts, bone marrow, and peripheral blood cells by Southern blot analysis. With PCR, analytical sensitivity was significantly increased, and deleted mtDNA was detected in all examined tissues from this patient. The patient had healthy parents and nine healthy siblings. No deleted mtDNA was detected in blood from the mother of the patient. The patient had an uneventful pregnancy and delivered at term. Deleted mtDNA could not be detected in placenta by Southern blot analysis. With PCR, deleted mtDNA was detected in the majority of placental specimens. This finding may, however, be due to contamination with maternal DNA. The patient's daughter was healthy at age 5 mo, and morphologic examination of muscle was normal. No transmission of deleted mtDNA to the daughter could be detected by Southern blot and PCR analysis of peripheral blood cells, bone marrow, fibroblasts, and muscle. The presence of deleted mtDNA was excluded at a fractional level of less than 1:100,000 in all examined tissues from the daughter.