Mitochondrial diseases

Mitochondrial diseases, and particularly mitochondrial myopathies or encephalomyopathies, have drawn increasing attention in the past decade. Initially defined by morphologic changes in muscle ("ragged red fibers" and ultrastructural abnormalities of mitochondria), mitochondrial encephalomyopathies can now be classified according to biochemical defects involving: (1) mitochondrial transport; (2) substrate oxidation; (3) Krebs cycle; (4) respiratory chain; and (5) oxidation-phosphorylation coupling. For each biochemical group of disorders, the authors describe clinical presentations and biochemical findings. These disorders are especially interesting from the genetic point of view because mitochondria have their own DNA (mtDNA), which encodes 13 polypeptides, all of them subunits of respiratory chain complexes. Other mitochondrial proteins are encoded by nuclear DNA, synthesized in the cytoplasm, and imported into the mitochondria by a complex mechanism. Because mtDNA is inherited strictly by maternal, cytoplasmic inheritance, mitochondrial diseases can be transmitted by Mendelian or by non-Mendelian, maternal inheritance, as illustrated by human pathology.     

Mitochondrial encephalomyopathies

Mitochondrial encephalomyopathies are diseases caused by defective oxidative phosphorylation (OXPHOS), and affect the nervous system and/or skeletal muscle. They have emerged as a major entity among the neurometabolic diseases of childhood with an incidence of 1 in 11,000 children, and also have a high prevalence in adults. The first pathogenic mutation of human mitochondrial DNA (mtDNA) was discovered in 1988. Since then more than 100 mutations of mtDNA have been reported, including point mutations of genes encoding transfer RNA, ribosomal RNA, and proteins, as well as large-scale deletions. The first nuclear-DNA gene mutation causing OXPHOS disease was described in 1995. Mutations in nuclear genes may affect the respiratory chain by various mechanisms. Pathogenic mutations of nuclear-DNA-encoded subunits of complex I and II have been demonstrated as have mutations of respiratory chain assembly proteins. Several nuclear genes associated with mtDNA maintenance have been found to be associated with mitochondrial disorders since mutations in these genes predispose to multiple mtDNA deletions and/or reduced copy number of mtDNA. The genotype-phenotype correlation is not yet entirely clear, but new animal models will enhance our ability to study the pathophysiology of OXPHOS disorders.     

Mitochondrial encephalomyopathies: an update

A genetic classification of the mitochondrial encephalomyopathies includes disorders due to defects of mitochondrial DNA (mtDNA) and disorders due to defects of nuclear DNA (nDNA). Recent progress in mtDNA-related diseases includes: (i) new pathogenic mutations in protein-coding genes, especially those encoding subunits of complex I (ND genes); (ii) the pathogenic nature of homoplasmic mutations, whose expression is regulated by environmental and genetic factors; (iii) increasing interest in the functional and pathophysiological role of haplotypes. Advances in mendelian mitochondrial diseases include: (i) new mutations in genes for complex I subunits; (ii) identification of new mutant ancillary proteins associated with complex IV and complex V deficiencies; (iii) better molecular understanding of disorders due to faulty intergenomic communication, which are associated with multiple mtDNA deletions, mtDNA depletion, or defects of mtDNA translation; (iv) the pathogenic role of alterations of the inner mitochondrial membrane phospholipid components, especially cardiolipin; (v) the emerging importance of defects in mitochondrial motility, fission, or fusion.     

Diseases of oxidative phosphorylation due to mtDNA mutations

Mitochondrial encephalomyopathies are disorders due to biochemical defects in the respiratory chain, which is under dual genetic control: 13 proteins are encoded by mitochondrial DNA (mtDNA), while all others are encoded by nuclear DNA. In the past 12 years, the small circle of mtDNA has proven to be a Pandora's box of pathogenic mutations, associated with a bewildering variety of multisystemic or tissue-specific disorders. After summarizing the principles of mitochondrial genetics, we attempt to provide general principles and practical clues to the diagnosis of mtDNA-related disorders by reviewing sequentially clinical presentation, family history, laboratory data, neuroradiology, exercise physiology, muscle morphology, muscle biochemistry, and molecular genetics.     

Surveyor nuclease detection of mutations and polymorphisms of mtDNA in children

Mitochondrial encephalomyopathies are complex disorders with wide range of clinical manifestations. Particularly time-consuming is the identification of mutations in mitochondrial DNA. A group of 20 children with clinical manifestations of mitochondrial encephalomyopathies was selected for molecular studies. The aims were (a) to identify mutations in mtDNA isolated from muscle and (b) to verify detected mutations in DNA isolated from blood, in order to assess the utility of a Surveyor nuclease assay kit for patient screening. The most common changes found were polymorphisms, including a few missense mutations altering the amino acid sequence of mitochondrial proteins. In two boys with MELAS (i.e., mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), a mutation A→G3243 was detected in the tRNALeu gene of mtDNA isolated from muscle and blood. In one boy, the carrier status of his mother was confirmed, based on molecular analysis of DNA isolated from blood. A method using Surveyor nuclease allows systematic screening for small mutations in mtDNA, using as its source blood of the patients and asymptomatic carriers. The method still requires confirmation studying a larger group. In some patients, the use of this method should precede and might limit indications for traumatic muscle and skin biopsy.     

Pathogenic mitochondrial DNA mutations in protein-coding genes

More than 200 disease-related mitochondrial DNA (mtDNA) point mutations have been reported in the Mitomap (http://www.mitomap.org) database. These mutations can be divided into two groups: mutations affecting mitochondrial protein synthesis, including mutations in tRNA and rRNA genes; and mutations in protein-encoding genes (mRNAs). This review focuses on mutations in mitochondrial genes that encode proteins. These mutations are involved in a broad spectrum of human diseases, including a variety of multisystem disorders as well as more tissue-specific diseases such as isolated myopathy and Leber hereditary optic neuropathy (LHON). Because the mitochondrial genome contains a large number of apparently neutral polymorphisms that have little pathogenic significance, along with secondary homoplasmic mutations that do not have primary disease-causing effect, the pathogenic role of all newly discovered mutations must be rigorously established. A scoring system has been applied to evaluate the pathogenicity of the mutations in mtDNA protein-encoding genes and to review the predominant clinical features and the molecular characteristics of mutations in each mtDNA-encoded respiratory chain complex.     

An A8296G mutation in the MT-TK gene of a patient with epilepsy - a disease-causing mutation or rare polymorphism?

Mitochondrial DNA (mtDNA) mutations are an important cause of human diseases. mtDNA could be considered a candidate modifying factor in neurodegenerative disorders. A homoplasmic A8296G mutation was detected in a 24-year-old patient with idiopathic generalized epilepsy. The A8296G mutation in the mitochondrial DNA MT-TK gene has been associated with severe mitochondrial diseases. The pathogenicity of this mutation or its association with a specific disease is unclear. This mutation has already been reported exclusively as well as together with other mutations during trials of mtDNA. As in this case, the mutation was homoplasmic and there were no clinical findings in other family members. We suggest that this mutation is a rare polymorphism or may be a pathogenic mutation in combination with other mutations outside of the MT-TK gene.     

Deletions of mitochondrial DNA in Kearns-Sayre syndrome

We have identified large-scale deletions in muscle mitochondrial DNA (mtDNA) in seven of seven patients with Kearns-Sayre syndrome (KSS). We found no detectable deletions in the mtDNA of ten non-KSS patients with other mitochondrial myopathies or encephalomyopathies, or three normal controls. The deletions ranged in size from 2.0 to 7.0 kb, and did not localize to any single region of the mitochondrial genome. The proportion of mutated genomes in each KSS patient ranged from 45% to 75% of total mtDNA. There was no correlation between the size or site of the deletion, biochemical abnormality of mitochondrial enzymes, or clinical severity. The data bolster arguments that KSS is a unique disorder and genetic in origin.     

Mitochondrial encephalomyopathies: biochemical approach.

Thanks to recent advances in the molecular genetics of mitochondrial encephalomyopathies, we can now begin to correlate genetic lesions with biochemical defects. In the fatal infantile myopathy due to cytochrome c oxidase (COX) deficiency, an autosomal recessive condition, immunocytochemical studies have shown an isolated defect of subunit VIIa, which is 1 of the only 2 tissue-specific subunits of human COX. In muscle biopsies from patients with Kearns-Sayre syndrome, a multisystem disorder characterized by deletions of the mitochondrial DNA (mtDNA), the activities of all mitochondrial enzymes containing mtDNA-encoded subunits are decreased. The results of Northern analysis, in situ hybridization, and immunocytochemistry in muscle, and of mitochondrial protein synthesis in cultured fibroblasts suggest that partially deleted mtDNAs are transcribed but not translated, probably due to lack of indispensable tRNAs.     

Content of mutant mitochondrial DNA and organ dysfunction in a patient with a MELAS subgroup of mitochondrial encephalomyopathies

A point mutation of mitochondrial tRNA^Leu(UUR) gene is responsible for a MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) subgroup of mitochondrial encephalomyopathies. In most cases, the mutant mitochondrial DNA (mtDNA) coexists with normal mtDNA in a heteroplasmic manner. In order to quantify the content of mutant mtDNA, we developed a quantitative method of PCR. Using this method, the distribution of the mutant mtDNA was examined in 32 different tissues among 18 autopsied organs from a patient with MELAS, who had shown hypophyseal dysfunction. The percentage of the mutant mtDNA at nucleotide number 3243 in each tissue was ranged between 22% and 95%. The content of the mutant mtDNA was at the highest (95%) in the hypophysis and higher in the cerebral cortex than in the white matter. This study shows a possible correlation of tissue dysfunction with accumulation of the mutant mtDNA within the brain.     

Lack of apoptosis in mitochondrial encephalomyopathies

BACKGROUND/OBJECTIVE: Apoptosis, or programmed cell death, is an evolutionary conserved mechanism essential for morphogenesis and tissue homeostasis, but it plays an important role also in pathologic conditions, including neurologic disorders. Its execution pathway is critically regulated at the mitochondrial level. Evidence of apoptosis in muscle specimens was investigated in patients with genetically defined mitochondrial encephalomyopathies. METHODS: Thirty-three muscle biopsies from patients with genotypically different mitochondrial diseases (single and multiple deletions, A3243G/A8344G point mutations of the mitochondrial DNA) were studied. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction was used as a marker of nuclear DNA fragmentation, as well as antibodies against pro- (Fas) or anti- (Bcl-2) apoptotic factors. Also, because one hallmark of apoptosis is morphologic, ultrastructural studies were performed on skeletal muscle from 18 of 33 patients, examining both phenotypically normal and ragged red fibers. RESULTS: In all muscle biopsies, no significant expression of either pro (Fas) and inhibiting (Bcl-2) apoptosis-related proteins was found, nor TUNEL positivity. This latter finding is confirmed by lack of morphologic evidence of apoptosis in all the fibers examined at the ultrastructural level. CONCLUSION: The authors' findings suggest that genetically determined defects of oxidative phosphorylation do not induce the apoptotic process and that apoptosis is not involved in the pathogenesis of mitochondrial disorders.     

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)).     

Neonatal mitochondrial encephaloneuromyopathy due to a defect of mitochondrial protein synthesis

Mitochondrial diseases are clinically and genetically heterogeneous disorders due to primary mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). We studied a male infant with severe congenital encephalopathy, peripheral neuropathy, and myopathy. The patient's lactic acidosis and biochemical defects of respiratory chain complexes I, III, and IV in muscle indicated that he had a mitochondrial disorder while parental consanguinity suggested autosomal recessive inheritance. Cultured fibroblasts from the patient showed a generalized defect of mitochondrial protein synthesis. Fusion of cells from the patient with 143B206 rho(0) cells devoid of mtDNA restored cytochrome c oxidase activity confirming the nDNA origin of the disease. Our studies indicate that the patient has a novel autosomal recessive defect of mitochondrial protein synthesis.     

Mitochondrial diseases

Since the first reports of disorders associated with mitochondrial DNA (mtDNA) defects more than a decade ago, the small mtDNA circle has been a Pandora's box of pathogenic mutations associated with human diseases. The "morbidity map" of mtDNA has gone from one point mutation and a few deletions in 1988 to more than 110 point mutations as of September, 2001. Nuclear DNA defects affecting mitochondrial function and mtDNA replication and integrity have also been identified in the past few years and more are expected. As a result, human "mitochondrial" diseases have evolved beyond the novelty diagnoses of a decade ago into an important area of medicine, and thus, the diagnostic principles of these disorders ought to be familiar to the clinician. In this article, the authors, we summarize the principles of mitochondrial genetics and discuss the common phenotypes, general diagnostic approach, and possible therapeutic venues for these fascinating disorders.     

The "S" in MELAS.

MELAS syndrome is one of several mitochondrial-inherited encephalomyopathies distinguished from the others by its unique stroke-like episodes. A case is presented that illustrates the importance of acknowledging the heteroplasmic nature of this disease when making its diagnosis. The mitochondrial DNA (mtDNA) point mutation characteristic of MELAS was eventually detected by analysis of a muscle biopsy specimen after initial studies of a serum sample were negative for the same genetic defect. Other diagnostic features of MELAS syndrome are described; these include characteristic computed tomography (CT), magnetic resonance imaging (MRI), angiography, single photon emission computed tomography using N-isopropyl-p-[123-I]-Iodoamphetamine (IMP-SPECT), and pathological findings. Finally, various theories regarding the etiology of stroke in MELAS syndrome as well as available treatment options are discussed.     

Neurological disorders due to mutations of the mitochondrial genome.

The rapidly expanding list of human diseases due to lesions of mitochondrial DNA includes myopathies, encephalopathies, cardiomyopathies, or various combinations of the latter, leading to multisystem disorders, which can also affect visceral organs. Five maternally inherited diseases, mainly affecting muscle and brain, are due to point mutations of mitochondrial genes encoding either respiratory chain polypeptides or transfer RNAs. On the other hand, three sporadic entities, Chronic Progressive External Ophthalmoplegia, Kearns-Sayre syndrome, and Pearson's pancreas-bone marrow syndrome, are due to single large-scale deletions of mitochondrial DNA. In addition, multiple deletions are the molecular hallmark of familial encephalomyopathies, inherited as either autosomal dominant or autosomal recessive traits. Finally, tissue-specific depletion of mitochondrial DNA was found in an autosomal recessive disease affecting either muscle, liver, kidney, or a combination of the three. Point mutations and slipped mispairing during, or impairment of, mitochondrial replication are likely mechanisms involved in the pathogenesis of these lesions.     

The Mitochondrial Genome And Human Mitochondrial Diseases

To date, more than 100 point mutations and several hundreds of structural rearrangements of mitochondrial DNA (mtDNA) are known too be connected with characteristic neuromuscular and other mitochondrial syndromes varying from those causing death at the neonatal stage to diseases with late ages of onset. The immediate cause of mitochondrial disorders is a defective oxidative phosphorylation. Wide phenotypic variation and the heteroplasmy phenomenon, which some authors include in mutation load, are characteristic of human mitochondrial diseases. As the numbers of cases identified and pedigrees described increase, data on the genotype-phenotype interaction and the structure and frequency of pathogenic and conditionally pathogenic mtDNA mutations in human populations are rapidly accumulated. The data on the genetics and epidemiology of mitochondrial diseases are not only important for differential diagnosis and genetic counseling. Since both neutral and mildly pathogenic mutations of mtDNA are progressively accumulated in maternal phyletic lines, molecular analysis of these mutations permits not only reconstruction of the genealogical tree of modern humans, but also estimation of the role that these mutations play in natural selection.     

Mitochondrial function and mitochondrial DNA in a series of 64 patients suspected of having mitochondrial myopathy

Biochemical results concerning 64 patients suspected of mitochondrial myopathies are presented. Four clinical groups were studied including 21 encephalomyopathies, 42 ocular myopathies, 8 isolated myopathies and 3 cardiomyopathies. In 26 cases, the coexistence of a normal mitochondrial DNA and a mutated mitochondrial DNA (heteroplasmy) was found (19 simple deletions, 4 multiple deletions and 3 punctual mutations) and all cases presented with ocular disorders (excepted 2 cases with MERRF). Furthermore, 1 complex I deficiency (1 ocular myopathy), 1 complex IV deficiency (1 adult encephalomyopathy type Leigh), 3 complexes I + IV deficiencies (2 cases with a cardiomyopathy and 1 familial MELAS) and 2 pyruvate (1 adult from of Leigh's encephalomyopathy) dehydrogenase deficiencies (clinically and genetically different) did not show evidence of mitochondrial DNA mutation.     

Normal insulin receptors in mitochondrial myopathies with ophthalmoplegia

Seven patients with histologically proven mitochondrial myopathy with ophthalmoplegia (OMM), 6 of them nondiabetic, 1 affected by diabetes mellitus (DM), were submitted to a study of glucose tolerance and of insulin receptors on peripheral mononuclear cells and cultured skin fibroblasts. The diabetic patient, who had the typical features of the Kearns-Sayre syndrome (KSS) and deleted muscle mitochondrial DNA (mtDNA) presented a low insulin secretion rate under physiological stimuli (intravenous glucose and glucagon) whereas the insulin receptor parameters were found normal. The other patients showed a normal glucose tolerance and normal insulin receptors. Our data support the hypothesis that insulin receptors are not involved in the pathogenesis of DM associated with mitochondrial encephalomyopathies, in contrast to other neuromuscular inherited disorders. The clinical and biological features of DM presented by our KSS patient show normal insulin receptor parameters in spite of a defective insulin secretion, possibly depending on mitochondrial dysfunction.     

The incidence of mitochondrial encephalomyopathies in childhood: clinical features and morphological, biochemical, and DNA anbormalities

In this study we present incidence, point prevalence, and mortality figures of mitochondrial encephalomyopathies in a population-based study of children from western Sweden. Through the screening of registers and review of medical records, we identified 32 patients under 16 years of age from the study population who were diagnosed between January 1, 1984, and December 31, 1998. The incidence of mitochondrial encephalomyopathies in preschool children (<6 years of age) was 1 out of 11,000. The preschool incidence of Leigh's syndrome was 1 out of 32,000, and the preschool incidences of both Alper's syndrome and infantile mitochondrial myopathy with cytochrome C oxidase deficiency were 1 out of 51,000. The point prevalence January 1, 1999) of mitochondrial encephalomyopathies in children under 16 years of age was 1 out of 21,000. The median survival for patients with infantile onset was until 12 years of age. We identified 4 cases with mitochondrial DNA point mutations, 2 cases with mitochondrial DNA deletions, and 2 cases with nuclear mutations in the SURF1 gene. We conclude that mitochondrial encephalomyopathies are relatively common neurometabolic disorders in childhood.