Sara Shanske

Mitochondrial DNA Deletions in Progressive External Ophthalmoplegia and Kearns-Sayre Syndrome

We investigated the correlations of deletions of mitochondrial DNA in skeletal muscle with clinical manifestations of mitochondrial myopathies, a group of disorders defined either by biochemical abnormalities of mitochondria or by morphologic changes causing a ragged red appearance of the muscle fibers histochemically. We performed genomic Southern blot analysis of muscle mitochondrial DNA from 123 patients with different mitochondrial myopathies or encephalomyopathies. Deletions were found in the mitochondrial DNA of 32 patients, all of whom had progressive external ophthalmoplegia. Some patients had only ocular myopathy, whereas others had Kearns-Sayre syndrome, a multisystem disorder characterized by ophthalmoplegia, pigmentary retinopathy, heart block, and cerebellar ataxia. The deletions ranged in size from 1.3 to 7.6 kilobases and were mapped to different sites in the mitochondrial DNA, but an identical 4.9-kilobase deletion was found in the same location in 11 patients. Biochemical analysis showed decreased activities of NADH dehydrogenase, rotenone-sensitive NADH-cytochrome c reductase, succinate-cytochrome c reductase, and cytochrome c oxidase, four enzymes of the mitochondrial respiratory chain containing subunits encoded by mitochondrial DNA. We conclude that deletions of muscle mitochondrial DNA are associated with ophthalmoplegia and may result in impaired mitochondrial function. However, the precise relation between clinical and biochemical phenotypes and deletions remains to be defined.

Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.

Mammalian cytochrome c oxidase (COX) catalyses the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane. Mitochondrial DNA (mtDNA) encodes three COX subunits (I–III) and nuclear DNA (nDNA) encodes ten. In addition, ancillary proteins are required for the correct assembly and function of COX (refs 2, 3, 4, 5, 6). Although pathogenic mutations in mtDNA-encoded COX subunits have been described, no mutations in the nDNA-encoded subunits have been uncovered in any mendelian-inherited COX deficiency disorder. In yeast, two related COX assembly genes, SCO1 and SCO2 (for synthesis of cytochrome c oxidase), enable subunits I and II to be incorporated into the holoprotein. Here we have identified mutations in the human homologue, SCO2, in three unrelated infants with a newly recognized fatal cardioencephalomyopathy and COX deficiency. Immunohistochemical studies implied that the enzymatic deficiency, which was most severe in cardiac and skeletal muscle, was due to the loss of mtDNA-encoded COX subunits. The clinical phenotype caused by mutations in human SCO2 differs from that caused by mutations in SURF1, the only other known COX assembly gene associated with a human disease, Leigh syndrome.

Atypical clinical presentations associated with the MELAS mutation at position 3243 of human mitochondrial DNA

Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is commonly associated with an A-->G transition at position 3243 of the mitochondrial DNA. To determine the diversity of clinical syndromes associated with this mutation, 91 patients with mitochondrial encephalomyopathies that did not conform to the MELAS phenotype were screened. Twenty one patients with the 3243 mutation, most of whom had progressive external ophthalmoplegia (PEO) were found. Clinical features did not distinguish PEO patients with the 3243 mutation from those with large-scale deletions of mtDNA. However, most cases with single large-scale mtDNA deletions were sporadic, whereas most patients with the 3243 mutation had affected maternal relatives. Histochemical studies of muscle showed that cytochrome c oxidase (COX) deficiency was more severe in patients with PEO than in patients with typical MELAS, even though PEO patients had a lower percentage of mutant genomes in muscle. These data imply that the 3243 mutation is a major cause of familial PEO, and suggests that the threshold number of mtDNAs harboring the 3243 mutation necessary to affect a particular tissue vary in different patients. The proportion of mutant genomes in combination with other, still undefined, tissue-specific modulating factors seem to determine the overall clinical syndrome.

Clinical features associated with the A → G transition at nucleotide 8344 of mtDNA (“MERRF mutation”)

We looked for the A → G transition at position 8344 of mtDNA in 150 patients, most of them with diagnosed or suspected mitochondrial disease, to assess the specificity of this mutation for the MERRF phenotype, to define the clinical spectrum associated with the mutation, and to study the relationship between percentage of mutation in muscle and clinical severity. Our results confirm the high correlation between the A → G transition at position 8344 and the MERRF syndrome, but they also show that this mutation can be associated with other phenotypes, including Leighs syndrome, myoclonus or myopathy with truncal lipomas, and proximal myopathy. The absence of the mutation in four typical MERRF patients suggests that other mutations in the tRNALys gene, or elsewhere in the mitochondrial DNA, can produce the same phenotype.

Genetic Counseling and Prenatal Diagnosis for the Mitochondrial DNA Mutations at Nucleotide 8993

Mitochondrial genetics is complicated by heteroplasmy, or mutant load, which may be from 1%-99%, and thus may produce a gene dosage-type effect. Limited data are available for genotype/phenotype correlations in disorders caused by mtDNA mutations; therefore, prenatal diagnosis for mtDNA mutations has been hindered by an inability to predict accurately the clinical severity expected from a mutant load measured in fetal tissue. After reviewing 44 published and 12 unpublished pedigrees, we considered the possibility of prenatal diagnosis for two common mtDNA mutations at nucleotide 8993. We related the severity of symptoms to the mutant load and predicted the clinical outcome of a given mutant load. We also used the available data to generate empirical recurrence risks for genetic counseling, which may be used in conjunction with prenatal diagnosis.

Molecular analysis of the muscle pathology associated with mitochondrial DNA deletions.

Large–scale deletions of mitochondrial DNA (mtDNA) are associated with a subgroup of mitochondrial encephalomyopathies. We studied seven patients with Kearns–Sayre syndrome or isolated ocular myopathy who harboured a sub–population of partially–deleted mitochondrial genomes in skeletal muscle. Variable cytochrome c oxidase (COX) deficiencies and reduction of mitochondrially–encoded polypeptides were found in affected muscle fibres, but while many COX–deficient fibres had increased levels of mutant mtDNA, they almost invariably had reduced levels of normal mtDNA. Our results suggest that a specific ratio between mutant and wild–type mitochondrial genomes is the most important determinant of a focal respiratory chain deficiency, even though absolute copy numbers may vary widely.

Molecular Genetic Heterogeneity of Myophosphorylase Deficiency (McArdle's Disease)

BACKGROUND AND METHODS Myophosphorylase deficiency (McArdles disease) is one of the most common causes of exercise intolerance, muscle cramps, and recurrent myoglobinuria. The myophosphorylase gene has been sequenced and assigned to chromosome 11, but the molecular basis of McArdles disease is not known. We sequenced complementary DNA in 4 patients and studied genomic DNA by restriction-endonuclease analysis in 40 patients with McArdles disease. RESULTS Sequence analysis revealed three distinct point mutations: the substitution of thymine for cytosine at codon 49 in exon 1, changing an encoded arginine to a stop codon; the substitution of adenine for guanine at codon 204 in exon 5, changing glycine to serine; and the substitution of cytosine for adenine at codon 542 in exon 14, changing lysine to threonine. Analysis of restriction-fragment-length polymorphisms of appropriate fragments of genomic DNA after amplification with the polymerase chain reaction showed that 18 patients were homozygous for the stop-codon mutation, 6 had different mutations in the two alleles (compound heterozygotes), and 11 were presumed to be compound heterozygotes for a known mutation and an unknown one; only 5 patients had none of the three mutations. All three mutations were present in various combinations in five members of a family in which transmission appeared to be autosomal dominant. CONCLUSIONS McArdles disease is genetically heterogeneous, but the most common mutation is the substitution of thymine for cytosine at codon 49. These results suggest that in about 90 percent of patients the diagnosis of McArdles disease can be made from a patients leukocytes, thus avoiding the need for muscle biopsy.

Fatal infantile cytochrome c oxidase deficiency Decrease of immunologically detectable enzyme in muscle

A 2-month-old boy had progressive generalized weakness, hypotonia, and respiratory insufficiency requiring assisted ventilation. At age 31/2 months, he started having seizures and recurrent pulmonary infections; he died at age 7 months. Serum lactate was chronically elevated, but there was no aminoaciduria. Histochemical and ultrastructural studies of muscle biopsies at ages 2 and 3 months showed excessive mitochondria, lipid, and glycogen; a third biopsy at 6 months showed marked increase in perimysial fibrous and fat tissue. Cytochrome c oxidase activity was 7% of normal in the first biopsy and undetectable in the others. Cytochrome spectra of mitochondria isolated from postmortem muscle showed complete lack of cytochrome aa3 Antibodies were obtained against cytochrome c oxidase purified from normal human heart. Immunotitration and enzyme-linked immunosorbent assay (ELISA) showed decreased immunologically reactive enzyme protein in the patients muscle, but SDS-PAGE electrophoresis of immunoprecipitates of muscle mitochondrial extracts showed the presence of all cytochrome c oxidase subunits. These data suggest that decreased synthesis of one or more subunits may result in markedly decreased concentration of electrophoretically normal complex IV in skeletal muscle.

Risk of developing a mitochondrial DNA deletion disorder

BACKGROUND Pathogenic mitochondrial DNA (mtDNA) mutations are found in at least one in 8000 individuals. No effective treatment for mtDNA disorders is available, making disease prevention important. Many patients with mtDNA disease harbour a single pathogenic mtDNA deletion, but the risk factors for new cases and disease recurrence are not known. METHODS We did a multicentre study of 226 families in which a single mtDNA deletion had been identified in the proband, including patients with chronic progressive external ophthalmoplegia, Kearns Sayre syndrome, or Pearsons syndrome. We studied the relation between maternal age and the risk of unaffected mothers having an affected child, and determined the recurrence risks among the siblings and offspring of affected individuals. FINDINGS We noted no relation between maternal age and the risk of unaffected mothers having children with an mtDNA deletion disorder. None of the 251 siblings of the index cases developed clinical features of mtDNA disease. Risk of recurrence among the offspring of affected women was 4.11% (95% CI 0.86-11.54, or one in 117 to one in nine births). Only one of the mothers who had an affected child had a duplication of mtDNA in skeletal muscle. INTERPRETATION Unlike nuclear chromosomal rearrangements, incidence of mtDNA deletion disorders does not increase with maternal age, and unaffected mothers are unlikely to have more than one affected child. Affected women were previously thought to have a negligible chance of having clinically affected offspring, but the actual risk is, on average, about one in 24 births.

A Stop-Codon Mutation in the Human mtDNA Cytochrome c Oxidase I Gene Disrupts the Functional Structure of Complex IV

We have identified a novel stop-codon mutation in the mtDNA of a young woman with a multisystem mitochondrial disorder. Histochemical analysis of a muscle-biopsy sample showed virtually absent cytochrome c oxidase (COX) stain, and biochemical studies confirmed an isolated reduction of COX activity. Sequence analysis of the mitochondrial-encoded COX-subunit genes identified a heteroplasmic G-->A transition at nucleotide position 6930 in the gene for subunit I (COX I). The mutation changes a glycine codon to a stop codon, resulting in a predicted loss of the last 170 amino acids (33%) of the polypeptide. The mutation was present in the patients muscle, myoblasts, and blood and was not detected in normal or disease controls. It was not detected in mtDNA from leukocytes of the patients mother, sister, and four maternal aunts. We studied the genetic, biochemical, and morphological characteristics of transmitochondrial cybrid cell lines, obtained by fusing of platelets from the patient with human cells lacking endogenous mtDNA (rho0 cells). There was a direct relationship between the proportion of mutant mtDNA and the biochemical defect. We also observed that the threshold for the phenotypic expression of this mutation was lower than that reported in mutations involving tRNA genes. We suggest that the G6930A mutation causes a disruption in the assembly of the respiratory-chain complex IV.