EFNS Task Force on Molecular Diagnosis of Neurologic Disorders: guidelines for the molecular diagnosis of inherited neurologic diseases. Second of two parts.
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Cerebrovascular Disorders↗
Biomedical subjects
M Zeviani
Publications and source records attributed to M Zeviani.
Mitochondrial disorders.
The most relevant contribution to the elucidation of the molecular basis of mitochondrial disorders has come from the discovery of an impressive and ever expanding number of mutations of mitochondrial DNA. However, known mutations of mtDNA only account for a fraction of all the mitochondrial disorders in both infants and adults. A number of recent clinical and molecular observations indicate that many syndromes are caused by abnormalities in nuclear genes related to oxidative phosphorylation. Nuclear genes encode hundreds of proteins involved in mitochondrial biogenesis and oxidative phosphorylation. Nevertheless, the identification of the nuclear genes responsible for oxidative phosphorylation-related disorders has proceeded at a much slower pace, compared with the discovery and characterization of mtDNA mutations. The reasons for such a gap are numerous, including the rarity of the syndromes, their genetic heterogeneity, and our ignorance of this nuclear gene repertoire in humans. This scenario is rapidly changing, thanks to the discovery of several oxidative phosphorylation-related human genes, and to the identification in some of them of mutations responsible for different clinical syndromes. In addition, animal models have recently been generated, which will offer the opportunity to understand better the pathogenesis of specific oxidative phosphorylation defects, and to test in a rational and controlled fashion therapeutic strategies for the treatment of these disorders.
Animals↗
A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome.
We report on a novel frameshift mutation in the mtDNA gene encoding cytochrome c oxidase (COX) subunit III. The proband is an 11-year-old girl with a negative family history and an apparently healthy younger brother. Since 4 years of age, she has developed a progressive spastic paraparesis associated with ophthalmoparesis and moderate mental retardation. The presence of severe lactic acidosis and Leigh-like lesions of putamina prompted us to perform muscle and skin biopsies. In both, a profound, isolated defect of COX was found by histochemical and biochemical assays. Sequence analysis of muscle mtDNA resulted in the identification of a virtually homoplasmic frameshift mutation in the COIII gene, due to the insertion of an extra C at nucleotide position 9537 of mtDNA. Although the 9537C(ins) does not impair transcription of COIII, no full-length COX III protein was detected in mtDNA translation assays in vivo. Western blot analysis of two-dimensional blue-native electrophoresis showed a reduction of specific crossreacting material and the accumulation of early-assembly intermediates of COX, whereas the fully assembled complex was absent. One of these intermediates had an electrophoretic mobility different from those seen in controls, suggesting the presence of a qualitative abnormality of COX assembly. Immunostaining with specific antibodies failed to detect the presence of several smaller subunits in the complex lacking COX III, in spite of the demonstration that these subunits were present in the crude mitochondrial fraction of patient's cultured fibroblasts. Taken together, the data indicate a role for COX III in the incorporation and maintenance of smaller COX subunits within the complex.
Base Sequence↗
Role of adenine nucleotide translocator 1 in mtDNA maintenance.
Autosomal dominant progressive external ophthalmoplegia is a rare human disease that shows a Mendelian inheritance pattern, but is characterized by large-scale mitochondrial DNA (mtDNA) deletions. We have identified two heterozygous missense mutations in the nuclear gene encoding the heart/skeletal muscle isoform of the adenine nucleotide translocator (ANT1) in five families and one sporadic patient. The familial mutation substitutes a proline for a highly conserved alanine at position 114 in the ANT1 protein. The analogous mutation in yeast caused a respiratory defect. These results indicate that ANT has a role in mtDNA maintenance and that a mitochondrial disease can be caused by a dominant mechanism.
Amino Acid Sequence↗
Decreased aminoacylation of mutant tRNAs in MELAS but not in MERRF patients.
Mutations in human mitochondrial tRNA genes are associated with a number of multisystemic disorders. Using an assay that combines tRNA oxidation and circularization we have determined the relative amounts and states of aminoacylation of mutant and wild-type tRNAs in tissue samples from patients with MELAS syndrome (mito- chondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes) and MERRF syndrome (myoclonus epilepsy with ragged red fibers), respectively. In most, but not all, biopsies from MELAS patients carrying the A3243G substitution in the mitochondrial tRNA(Leu(UUR))gene, the mutant tRNA is under-represented among processed and/or aminoacylated tRNAs. In contrast, in biopsies from MERRF patients harboring the A8344G substitution in the tRNA(Lys)gene neither the relative abundance nor the aminoacylation of the mutated tRNA is affected. Thus, whereas the A3243G mutation may contribute to the pathogenesis of MELAS by reducing the amount of aminoacylated tRNA(Leu), the A8344G mutation does not affect tRNA(Lys)function in the same way.
Acylation↗
Ultrastructural analysis of extraocular muscle in chronic progressive external ophthalmoplegia.
Extraocular muscles are primarily involved in many mitochondrial diseases, but no reports exist regarding the morphological appearance of the muscles in cases of long-standing ocular myopathies. For this reason, muscle samples obtained from surgery in a sporadic case of chronic progressive external ophthalmoplegia (CPEO) were used for ultrastructural investigation and molecular analysis of mitochondrial DNA. Genetic testing revealed a heteroplasmic macrodeletion of about 5.0 kilobases in length, localized between the 9570- and 14619-base pair regions. Electron microscopy revealed focal areas of both disruption and abnormality of mitochondria in only some of the muscle fibers, producing "selective vacuolization." This ultrastructural pattern was highly selective and limited to some extraocular muscle fibers, sparing all the others. The "selective damage" observed in this case of CPEO resembles that case occurring in another mitochondrial disease, Leber hereditary optic neuropathy, where damage occurs only in the papillomacular bundle of the retina, sparing peripheral axons. It is possible that some anatomical and physiological factors play a leading role in both Leber hereditary optic neuropathy and ocular myopathies. The ultrastructural aspect herein observed needs to be further investigated to better understand whether a particular muscle fiber type is the target of mitochondrial impairment in CPEO.
Base Sequence↗
Separation of intact pyruvate dehydrogenase complex using blue native agarose gel electrophoresis.
We show that the blue native gel polyacrylamide electrophoresis system (BN-PAGE) can be applied to pyruvate dehydrogenase complex (PDC). BN-PAGE has been used extensively to study the multisubunit enzymes of oxidative phosphorylation, as nondenaturing separation in the first dimension maintains holoenzyme integrity. However, the standard protocol was inappropriate for PDC as, at 10 MDa, it is approximately ten times larger than the largest respiratory chain enzyme complex. Therefore, agarose was substituted for polyacrylamide. Moreover, a substantial decrease in salt concentration was necessary to prevent dissociation of PDC. As with standard BN-PAGE, immunoblots of second-dimensional sodium dodecyl sulfate-PAGE (SDS-PAGE) provided more detailed information on specific subunits and subcomplexes. The method was applied to human heart mitochondrial fragments, control cultured human cells, rho0 cells that lack mitochondrial DNA, and two cell lines derived from patients with PDC deficiency. The PDC deficient cell lines showed a clear correlation between amount of PDC holoenzyme and disease severity. In cells lacking mitochondrial DNA, synthesis and assembly of all PDC subunits (all nuclearly encoded) appeared normal, suggesting that respiratory function has no regulatory role in PDC biogenesis. Blue native agarose gel electrophoresis coupled with standard second-dimensional SDS-PAGE provides a new tool to be used in conjunction with biochemical assays and immunoblots of one-dimensional SDS-PAGE to further elucidate the nature of PDC in normal and disease states. Furthermore, other cellular protein complexes of 1 MDa or more can be analysed by this method.
Electrophoresis, Agar Gel↗
Neuromuscular syndrome associated with the 3291T-->C mutation of mitochondrial DNA: a second case.
A mutation was found in an Italian child affecting the gene encoding the mitochondrial transfer RNA for leucine (codon UUR). This mutation (3291T-->C) had previously been reported in a single Japanese patient. In contrast with the original patient, who suffered from early-onset mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), our patient presented an apparently isolated mild myopathy. Mutational analysis in the proband and her family showed that the mutation was heteroplasmic, and that its relative amount was positively correlated with the severity of the phenotype. These findings lead to the definitive confirmation that the 3291T-->C is indeed pathogenic. As commonly found in mitochondrial-DNA related disorders, also for this mutation different clinical manifestations can be associated with the same genetic abnormality.
Biopsy, Needle↗
A double mutation (A8296G and G8363A) in the mitochondrial DNA tRNA (Lys) gene associated with myoclonus epilepsy with ragged-red fibers.
OBJECTIVE: To define potential pathogenic mitochondrial DNA (mtDNA) point mutations in a patient with myoclonus epilepsy with ragged-red fibers (MERRF) syndrome. BACKGROUND: MERRF syndrome is typically associated with point mutations in the mtDNA tRNALys gene. METHODS: We performed morphologic, biochemical, and genetic analysis of muscle samples from the patient and four relatives. Molecular genetic studies included sequencing, PCR, and restriction enzyme analysis on whole muscle, blood, and single muscle fibers. RESULTS: Muscle biopsy showed cytochrome c oxidase (COX), negative ragged-red fibers (RRF), and a defect of complex I of the mitochondrial respiratory chain. We found an A8296G transition and a G8363A mutation in the mtDNA tRNALYs gene. The A8296G was almost homoplasmic in muscle and blood from the propositus and his oligosymptomatic maternal relatives. The G8363A mutation was heteroplasmic and more abundant in muscle than in blood, and its proportion correlated with clinical severity. Single muscle fiber analysis showed significantly higher levels of G8363A genomes in COX-negative than in normal fibers, and almost homoplasmic levels of mutant A8296G mtDNA in both COX-negative and normal fibers. The two mutations affect highly conserved nucleotides and were not found in controls. CONCLUSIONS: The G8363A mutation is pathogenic; the co-occurrence of the A8296G mutation is of unclear significance and is likely to be a rare polymorphism.
Adult↗
Loss-of-function mutations of SURF-1 are specifically associated with Leigh syndrome with cytochrome c oxidase deficiency.
Mutations of SURF-1, a gene located on chromosome 9q34, have recently been identified in patients affected by Leigh syndrome (LS), associated with deficiency of cytochrome c oxidase (COX), the terminal component of the mitochondrial respiratory chain. To investigate to what extent SURF-1 is responsible for human disorders because of COX deficiency, we undertook sequence analysis of the SURF-1 gene in 46 unrelated patients. We analyzed 24 COX-defective patients classified as having typical Leigh syndrome (LS(COX)), 6 patients classified as Leigh-like (LL(COX)) cases, and 16 patients classified as non-LS(COX) cases. Frameshift, stop, and splice mutations of SURF-1 were detected in 18 of 24 (75%) of the LS(COX) cases. No mutations were found in the LL(COX) and non-LS(COX) group of patients. Rescue of the COX phenotype was observed in transfected cells from patients harboring SURF-1 mutations, but not in transfected cell lines from 2 patients in whom no mutations were detected by sequence analysis. Loss of function of SURF-1 protein is specifically associated with LS(COX), although a proportion of LS(COX) cases must be the result of abnormalities in genes other than SURF-1. SURF-1 is the first nuclear gene to be consistently mutated in a major category of respiratory chain defects. DNA analysis can now be used to accurately diagnose LS(COX), a common subtype of Leigh syndrome.
Child, Preschool↗
Nuclear gene defects in mitochondrial disorders.
An increasing number of nuclear genes have been associated with abnormalities of oxidative phosphorylation and mitochondrial disorders. The protein products of these genes can be grouped into three categories: structural components of the respiratory chain, factors influencing the structural integrity or the copy number of mitochondrial DNA, and proteins which control the formation, assembly and turnover of the respiratory complexes. Loss-of-function mutations in SURF-1, a gene belonging to the third category, have been associated with Leigh syndrome with cytochrome c oxidase deficiency. Mature Surf-1 protein (Surf-1p) is a 30 kDa hydrophobic polypeptide whose function is still unknown. Using antibodies against human Surf-1p, we demonstrated that this protein is imported into mitochondria as a larger precursor. The same analysis revealed that no protein is present in cell lines harboring loss-of-function mutations of SURF-1, regardless of their type and position. We also generated several constructs with truncated or partially deleted SURF-1 cDNAs. None of these constructs, expressed into SURF-1 null mutant cells, were able to rescue the COX phenotype, suggesting that different regions of the protein are all essential for function. Finally, experiments based on 2D gel electrophoresis indicated that assembly of COX in SURF-1 null mutants is blocked at an early step, most likely before the incorporation of subunit II in the nascent intermediates composed of subunit I alone or subunit I plus subunit IV.
Chromosomes, Human↗
The mitochondrial DNA C3303T mutation can cause cardiomyopathy and/or skeletal myopathy.
OBJECTIVE: Several mutations in mitochondrial DNA have been associated with infantile cardiomyopathy, including a C3303T mutation in the mitochondrial transfer RNA(Leu(UUR)) gene. Although this mutation satisfied generally accepted criteria for pathogenicity, its causative role remained to be confirmed in more families. Our objective was to establish the frequency of the C3303T mutation and to define its clinical presentation. STUDY DESIGN: Families with cardiomyopathy and maternal inheritance were studied by polymerase chain reaction/restriction fragment length polymorphism analysis looking for the C3303T mutation. RESULTS: We found the C3303T mutation in 8 patients from 4 unrelated families. In one, the clinical presentation was infantile cardiomyopathy; in the second family, proximal limb and neck weakness dominated the clinical picture for the first 10 years of life, when cardiac dysfunction became apparent; in the third family, 2 individuals presented with isolated skeletal myopathy and 2 others with skeletal myopathy and cardiomyopathy; in the fourth family, one patient had fatal infantile cardiomyopathy and the other had a combination of skeletal myopathy and cardiomyopathy. CONCLUSIONS: Our findings confirm the pathogenicity of the C3303T mutation and suggest that this mutation may not be rare. The C3303T mutation should be considered in the differential diagnosis of skeletal myopathies and cardiomyopathy, especially when onset is in infancy.
Adolescent↗
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.
Adenine↗
Hearing impairment and neurological dysfunction associated with a mutation in the mitochondrial tRNASer(UCN) gene.
We studied a large Dutch family with maternally inherited, progressive, sensorineural hearing loss in 27 patients. Only in a single family member was the hearing loss accompanied by neurological symptoms including ataxia and dysarthria. DNA analysis of the mitochondrial genome revealed the insertion of a C at nucleotide position 7472 in the tRNASer(UCN) gene (7472insC mutation). We determined the percentage of mutant DNA (heteroplasmy) in blood from all family members, and found no correlation between hearing loss and leucocyte heteroplasmy. The 7472insC mutation was previously identified in a smaller family from Sicily with sensorineural hearing loss in 9 family members, six of them also presenting neurologically with ataxia and myoclonus. The presence of the 7472insC mutation in two different pedigrees strongly supports its pathogenicity. However, the interfamilial difference in penetrance of the neurologic abnormalities is most likely to be strongly influenced by secondary factors different from the 7472insC mutation, as heteroplasmy or age of the patients were similar in both families. This mutation should therefore be analysed in families with maternally inherited hearing loss, irrespective of whether the hearing loss is non-syndromic or accompanied by neurologic abnormalities.
Aminoglycosides↗
A third locus predisposing to multiple deletions of mtDNA in autosomal dominant progressive external ophthalmoplegia.
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Adult↗
Molecular phenotype of the np 7472 deafness-associated mitochondrial mutation in osteosarcoma cell cybrids.
The nucleotide pair (np) 7472 insC mitochondrial DNA mutation in the tRNA(Ser)(UCN) gene is associated with sensorineural deafness, combined in some individuals with a wider syndrome including ataxia and myo-clonus. Previous studies in osteosarcoma cell cybrids revealed only a mild respiratory defect linked to the mutation. We have investigated the biochemical and molecular consequences of the mutation, using a panel of seven osteosarcoma cell cybrids containing 100% mutant mtDNA, plus two cybrids carrying 100% wild-type mtDNA from the same patient. The mutation is associated with a mild growth deficit in selective (galactose) medium that is only significant in combination with a reduced mtDNA copy number, suggesting a mechanism that might modulate clinical phenotype. The mutation results in a 65% drop in the steady-state level of tRNA(Ser)(UCN), but causes at most only a very mild and quantitative abnormality of mitochondrial protein synthesis, associated with modest hypersensitivity to doxycyclin. No evidence for a specific defect in aminoacylation was obtained, and unlike the case with the np 7445 mutation, the pattern of RNA processing of light strand transcripts of the ND6 region was not systematically altered. Comparing the np 7472 and np 7445 mutant phenotypes in cultured cells suggests that sensorineural deafness can result from a functional insufficiency of mitochondrial tRNA(Ser)(UCN), to which some cells of the auditory system are especially vulnerable.
Base Sequence↗
Characterization of SURF-1 expression and Surf-1p function in normal and disease conditions.
Loss-of-function mutations of the SURF-1 gene have been associated with Leigh syndrome with cytochrome c oxidase (COX) deficiency. Mature Surf-1 protein (Surf-1p) is a 30 kDa hydrophobic polypeptide whose function is still unknown. Using antibodies against a recombinant, hemagglutinin-tagged Surf-1p, we have demonstrated that this protein is imported into mitochondria as a larger precursor, which is then processed into the mature product by cleaving off an N-terminal leader polypeptide of approximately 40 amino acids. By using western blot analysis with specific antibodies, we showed that Surf-1p is localized in and tightly bound to the mitochondrial inner membrane. The same analysis revealed that no protein is present in cell lines harboring loss-of-function mutations of SURF-1, regardless of their type and position. Northern blot analysis showed the virtual absence of specific SURF-1 transcripts in different mutant cell lines. This result suggests that several mutations of SURF-1 are associated with severe mRNA instability. To understand better whether and which domains of the protein are essential for function, we generated several constructs with truncated or partially deleted SURF-1 cDNAs. None of these constructs, expressed into Surf-1p null mutant cells, were able to rescue the COX phenotype, suggesting that different regions of the protein are all essential for function. Finally, experiments based on blue native two-dimensional gel electrophoresis indicated that assembly of COX in Surf-1p null mutants is blocked at an early step, most likely before the incorporation of subunit II in the nascent intermediates composed of subunit I alone or subunit I plus subunit IV. However, detection of residual amounts of fully assembled complex suggests a certain degree of redundancy of this system.
Animals↗
Leigh syndrome transmitted by uniparental disomy of chromosome 9.
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Aneuploidy↗