Biomedical subjects

M Zeviani

Publications and source records attributed to M Zeviani.

At least 37 records · Page 2Linked to original sources

PubMed1995

Sequence analysis of mitochondrial DNA in a new maternally inherited encephalomyopathy.

A heteroplasmic insertion of a 9-bp tandem repeat element was detected in the mitochondrial DNA of the maternal members of a large family. The mutation was contained within the non-coding region between the genes specifying subunit II of cytochrome c oxidase and tR-NA(Lys). The proband and most of his maternal relatives were affected by a late-onset mitochondrial encephalomyopathy of variable severity, characterized by a unique combination of symptoms. Extensive screening of a large series of DNA samples, collected from unrelated normal individuals as well as patients affected by different neurological disorders, consistently failed to detect the 9-bp insertion, with two exceptions: a patient suffering from a syndrome virtually identical to that described in our original family and a child affected by bilateral striatal necrosis, a disorder which has been attributed to impairment of mitochondrial oxidative phosphorylation. These considerations suggest that the 9-bp insertion is pathogenic and that the region affected by the mutation may play a previously unsuspected functional role in mtDNA gene expression.

Aged↗

PubMed1995

Early-onset encephalomyopathy associated with tissue-specific mitochondrial DNA depletion: a morphological, biochemical and molecular-genetic study.

A male infant, born from consanguineous parents, suffered from birth with a progressive neuromuscular disorder characterized by psychomotor delay, hypotonia, muscle weakness and wasting, deep-tendon areflexia and spastic posture. High levels of lactic acid in blood and cerebrospinal fluid suggested a mitochondrial respiratory chain defect. Muscle biopsy revealed ragged-red and cytochrome c oxidase-negative fibres, lipid accumulation and dystrophic changes. Multiple defects of respiratory complexes were detected in muscle homogenate, but cultured fibroblasts, myoblasts and myotubes were normal. Southern blot analysis showed markedly reduced levels of mitochondrial DNA (mtDNA) in muscle, while lymphocytes, fibroblasts and muscle precursor cells were normal. Neither depletion of mtDNA nor abnormalities of the respiratory complexes were observed in innervated muscle fibres cultured for as long as 4 months. No mutations were observed in two candidate nuclear genes, mtTFA and mtSSB, retro-transcribed, amplified and sequenced from the proband's mRNA. Sequence analysis of the mtDNA D-loop and of the origin of replication of the mtDNA light strand failed to identify potentially pathogenic mutations of these replicative elements in the proband's muscle mtDNA. Our findings indicate that mtDNA depletion is due to a nuclear encoded gene and suggest that the abnormality underlying defective mtDNA propagation must occur after muscle differentiation in vivo.

Age of Onset↗

PubMed1995

Genotype to phenotype correlations in mitochondrial encephalomyopathies associated with the A3243G mutation of mitochondrial DNA.

We studied 22 subjects carrying the A3243G point mutation of human mitochondrial DNA (mtDNA). In 14 cases the clinical phenotype was characterized by mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), while 8 patients had chronic progressive external ophthalmoplegia (CPEO). The proportion of A3243G heteroplasmy in muscle was determined by two methods; densitometry on a diagnostic restriction-fragment length polymorphism and solid-phase mini-sequencing. We found a highly significant inverse correlation between the percentage of A3243G mutation and the specific activity of complex I, the respiratory complex with the highest number of mtDNA-encoded subunits, suggesting a direct effect of the mutation on mtDNA translation. No correlation was observed between the percentage of mutated mtDNA and the presence or absence of specific clinical features, such as stroke, ophthalmoplegia and diabetes mellitus. However, in the MELAS group the percentage of mutated mtDNA molecules was strongly correlated with the age of onset, while no such correlation was found in the CPEO group, suggesting a different time-dependent evolution of the mutation in the two groups. Finally, in contrast with other mtDNA mutations associated with ragged-red fibres (RRF), in both MELAS3243 and CPEO3243 we observed a high proportion of RRF that were positive to the histochemical reaction to cytochrome c oxidase, a morphological feature that seems to be specific for the neuromuscular phenotypes associated with mutations affecting the tRNA(Leu(UUR)) gene.

Adolescent↗

PubMed1995

Epilepsia partialis continua associated with NADH-coenzyme Q reductase deficiency.

We report the clinical, neuroradiological and biochemical features of a patient with epilepsia partialis continua (EPC). MRI studies disclosed multiple cortico-subcortical areas of abnormal signal intensity. The activity of complex I of the mitochondrial respiratory chain was markedly reduced in skeletal muscle. The biochemical defect was reflected in vivo by a failure of brain and skeletal muscle bioenergetics, as shown by exercise and phosphorus magnetic resonance spectroscopy (31P-MRS) studies. Muscle morphology was repeatedly normal, and molecular genetic analysis of mitochondrial DNA was not informative. On the basis of in vivo and in vitro findings, the observed defect of the mitochondrial respiratory chain was considered the underlying biochemical pathogenesis of the disease. The observation of an oxidative defect in the brain and skeletal muscle of a patient with EPC emphasizes the importance of studying mitochondrial energy metabolism in patients with EPC not associated with primary CNS lesions when clinical and morphological findings suggesting a mitochondrial disorder are lacking. 31P-MRS can be a useful method to uncover deficits of CNS mitochondrial function and provide the indication for further biochemical studies.

Adult↗

PubMed1995

An autosomal locus predisposing to deletions of mitochondrial DNA.

The molecular mechanisms by which the nuclear genome regulates the biosynthesis of mitochondrial DNA (mtDNA) are only beginning to be unravelled. A naturally occurring in vivo model for a defect in this cross-talk of two physically separate genomes is a human disease, an autosomal dominant progressive external ophthalmoplegia, in which multiple deletions of mtDNA accumulate in the patients' tissues. The assignment of this disease locus to 10q 23.3-24.3 is the first direct evidence for involvement of both nuclear and mitochondrial genomes in a single disorder.

Base Sequence↗

PubMed1995

Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh's syndrome: genetic evidence based on patient's-derived rho degrees transformants.

Defects of the respiratory chain carrying out oxidative phosphorylation (OXPHOS) are the biochemical hallmark of human mitochondrial disorders. Faulty OXPHOS can be due to mutations in either nuclear or mitochondrial genes, that are involved in the synthesis of individual respiratory subunits or in their post-translational control. The most common mitochondrial disorder of infancy and childhood is Leigh's syndrome, a severe encephalopathy, often associated with a defect of cytochrome c oxidase (COX). In order to demonstrate which genome is primarily involved in COX-deficient (COX(-))-Leigh's syndrome, we generated two lines of transmitochondrial cybrids. The first was obtained by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts, with mitochondrial DNA-less (rho degree) transformant fibroblasts derived from a patient with COX(-))-Leigh's syndrome. The second cybrid line was obtained by fusing rho degree cells derived from 143B.TK- human osteosarcoma cells, with cytoplasts derived from the same patient. The first cybrid line showed a specific and severe COX(-) phenotype, while in the second all the respiratory chain complexes, including COX, were normal. These results indicate that the COX defect in our patient is due to a mutation of a nuclear gene. The use of cybrids obtained from 'customized', patient-derived rho degree cells can have wide applications in the identification of respiratory chain defects originated by nuclear DNA-encoded mutations, and in the study of nuclear DNA-mitochondrial DNA interactions.

Cell Line↗

PubMed1995

Maternally inherited hearing loss, ataxia and myoclonus associated with a novel point mutation in mitochondrial tRNASer(UCN) gene.

We report on a new maternally-inherited syndrome characterized by a combination of sensorineural hearing loss, ataxia and myoclonus in a large kindred from Sicily. Hearing loss was the most widespread and sometimes the only symptom found in family members. Sequence analysis of the mitochondrial DNA regions encompassing the tRNA genes revealed the presence of a heteroplasmic insertion at nucleotide position 7472. The insertion adds a seventh cytosine to a six-cytosine run that is part of the mitochondrial tRNASer(UCN) gene. Conformational analysis showed that this mutation is likely to alter the structure of the T psi C loop in the tRNASer(UCN) clover leaf secondary structure. Moreover, the degree of heteroplasmy in blood and muscle was correlated with the clinical phenotype, and homoplasmic mutant hybrids showed decreased complex I activity, low oxygen consumption and high lactic acid output, indicating faulty oxidative phosphorylation. Finally, mutation was absent in 381 unrelated maternal lineages, suggesting specific segregation with the disease. We propose that the C7472 insertion-mutation is pathogenic, and etiologically related to hearing loss and other symptoms that define a novel maternally-inherited clinical entity.

Ataxia↗

PubMed1995

Evidence of linkage between susceptibility to multiple sclerosis and HLA-class II loci in Italian multiplex families.

To verify whether multiallelic polymorphisms belonging to HLA class II genes are linked to multiple sclerosis (MS) in the Italian population, we studied 28 multiplex MS families originating from different areas of Italy. Allelic characterization was carried out by analysis of RFLPs and oligonucleotide typing. Evidence supporting the existence of linkage between MS susceptibility and the HLA class II loci DRB1, DQA1 and DQB1 was provided using two non-parametric tests, affected sib-pair analysis, and affected-pedigree-member (APM) analysis. The APM analysis also suggested the existence of genetic heterogeneity for the HLA class II loci and MS susceptibility in our series. Linkage disequilibrium between MS susceptibility and the haplotype DRB1*1501,DQA1*0102,DQB1*0602 was demonstrated by applying the transmission linkage disequilibrium test to our families. Finally, lod score analysis suggests that in our Italian families, MS susceptibility is conferred by HLA class II alleles according to a low-penetrance autosomal recessive mode of inheritance.

Alleles↗

PubMed1994

Order of six loci at 2q24-q31 and orientation of the HOXD locus.

HOXD, a gene cluster of 9 homeobox genes of the Antennapedia class; EVX2, a homeobox gene related to Drosophila-even-skipped gene; DLX1 and DLX2, two homeobox genes related to the Drosophila distal-less gene; and TTN and NEB, the genes for the two giant molecules titin and nebulin, both involved in the sarcomere structure, have been previously mapped to human 2q31-q32 and to mouse chromosome 2. We studied their relative order in human by applying FISH to three balanced chromosome rearrangements each with a breakpoint at 2q31. Unambiguous results led us to map these genes and to orient the HOXD locus along chromosome 2 according to the following order: cen, NEB, DLX1-DLX2, EVX2, HOXD (5'-3'), TTN, tel. All of these genes are part of a syntenic region covering 5-10 cM and conserved since the divergence of humans and rodents, and thus the same loci order should be present in mouse. FISH in metaphases of approximately 500 bands localized NEB to 2q24.1-q24.2, while HOXD and TTN were localized to 2q31.

Animals↗

PubMed1994

De novo and inherited deletions of the 5q13 region in spinal muscular atrophies.

Spinal muscular atrophies (SMAs) represent the second most common fatal autosomal recessive disorder after cystic fibrosis. Childhood spinal muscular atrophies are divided into severe (type I) and mild forms (types II and III). By a combination of genetic and physical mapping, a yeast artificial chromosome contig of the 5q13 region spanning the disease locus was constructed that showed the presence of low copy repeats in this region. Allele segregation was analyzed at the closest genetic loci detected by markers C212 and C272 in 201 SMA families. Inherited and de novo deletions were observed in nine unrelated SMA patients. Moreover, deletions were strongly suggested in at least 18 percent of SMA type I patients by the observation of marked heterozygosity deficiency for the loci studied. These results indicate that deletion events are statistically associated with the severe form of spinal muscular atrophy.

Alleles↗

PubMed1994

Single-stranded-DNA-binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties.

The gene for the mature human mitochondrial single-stranded-DNA binding protein (HsmtSSB) has been transferred into a protein-overproducing vector and expressed in Escherichia coli. The protein was purified to homogeneity and its physicochemical properties were investigated. From sequence comparison, HsmtSSB shows some similarities to the N-terminal part of the single-stranded DNA-binding protein (SSB) from E. coli (EcoSSB). Hydrodynamic measurements show the protein to be tetrameric and give a sedimentation coefficient of 4.1 S corresponding to a C-terminally shortened EcoSSB. Electron-microscopic images of the free protein show a globular tetrahedral structure. Binding of poly(desoxythymidylic acid) [poly(dT)] leads to a reduction of the tryptophan fluorescence of the protein up to 96%. Fluorescence titrations with poly(dT) show apparent binding-site sizes of 50-70 nucleotides/tetramer between 0.05 M and 2 M NaCl. Binding to poly(dT) proceeds in a nearly diffusion-controlled reaction with an association-rate constant kass of 4 x 10(8) M-1s-1. The rate-limiting step is the formation of a transient complex where less than four binding sites on the protein are involved and the reshuffling of the protein on the linear matrix is fast. Electron microscopy of the complex with poly(dT) using negative staining shows a nearly random distribution of the protein between the individual poly(dT) strands. This leads to the conclusion that the binding cooperativity is low (omega < 150). The two tryptophans of HsmtSSB were replaced by threonine and tyrosine. The environment of both residues is influenced by nucleic acid binding with mutations of Trp68 strongly reducing the DNA-binding affinity of the protein.

Binding Sites↗

PubMed1994

Mitochondrial myopathy: correlation between oxidative defect and mitochondrial DNA deletions at single fiber level.

In situ hybridization combined with immunohistochemical techniques has been applied to study patients affected by mitochondrial myopathies with large mitochondrial (mt)DNA deletions. All patients' muscle biopsies showed ragged red fibers (RRFs) and cytochrome oxidase (COX) deficiency. Two digoxigenin-labeled, polymerase chain reaction (PCR)-amplified DNAs were used as probes. One probe was designed to hybridize only with wild-type mtDNAs, while the other recognized both wild-type and deleted mtDNAs. Concomitant immunocytochemical analysis using antibodies against subunits II, III, (encoded by mtDNA) and IV (encoded by nuclear DNA) of COX was carried out. In our patients deleted mtDNAs are overexpressed in COX-negative RRFs, while wild-type mtDNAs are decreased in the same fibers. Immunohistochemistry studies show that COX IV is overexpressed in RRFs and that COX II and COX III subunits are still present. Deleted mtDNAs are spatially segregated in muscle fibers, where they interfere with the local population of normal mitochondrial genomes, causing a regional deficiency of the mitochondrial respiratory activity.

Adolescent↗

PubMed1994

The myelin basic protein gene is not a major susceptibility locus for multiple sclerosis in Italian patients.

To verify whether multiallelic polymorphism adjacent to the gene encoding for myelin basic protein is associated with or linked to multiple sclerosis in Italians, we studied 54 sporadic patients, 55 control subjects and 18 families with two or more affected individuals. Allelic typing was carried out by analysis of fragment length polymorphisms after DNA amplification by the polymerase chain reaction. The presence of linkage with the disease was tested according to either autosomal dominant or autosomal recessive modes of inheritance, and with or without the introduction of liability classes accounting for the age of the individuals. Furthermore sib-pair analysis was performed in 11 siblings. No evidence for association or linkage between the myelin basic protein gene polymorphism and multiple sclerosis was found. Our data indicate that in the Italian population the myelin basic protein gene does not play a major role in conferring genetic susceptibility to multiple sclerosis, and suggest that the latter is a heterogeneous phenomenon, possibly influenced by the different ethnic origin of the populations which have been investigated.

Adult↗

PubMed1994

Mitochondrial myopathies.

Major new advances in the genetic and biochemical characterization of mitochondrial myopathies are discussed, within a general presentation of this important new area of human pathology. Mitochondrial disorders can be due to mutations in either nuclear or mitochondrial genes involved in the synthesis of individual respiratory chain subunits or in their posttranslational control. Although no mutations of nuclear-encoded oxidative phosphorylation subunits have been reported so far in humans, numerous biochemically defined disorders are attributed to nuclear gene defects. In contrast, molecular lesions of mitochondrial DNA are recognized as an increasingly frequent cause of defective oxidative phosphorylation. Numerous new mutations recently have been identified, including both maternally inherited point mutations and sporadic large-scale rearrangements. In addition, the identification of new or overlap syndromes has substantially broadened the clinical spectrum of mitochondrial disorders. To gain insight into the pathogenesis of these disorders, the relationship between specific clinical presentations and the mitochondrial genotype has been intensively investigated. In most cases, the phenotypic expression of the mitochondrial DNA mutations depends on the interplay among the relative amount of mutated vs wild-type genomes, ie, the degree of mitochondrial heteroplasmy and its tissue and cell distribution, the reliance of the affected tissues on aerobic energy supply, the age and gender of the individual, and other still poorly understood factors including individual "nuclear genetic background" and environmental factors.

Cell Nucleus↗

PubMed1994

Defective respiratory capacity and mitochondrial protein synthesis in transformant cybrids harboring the tRNA(Leu(UUR)) mutation associated with maternally inherited myopathy and cardiomyopathy.

We studied the physiometabolic effects of a mitochondrial DNA (mtDNA) heteroplasmic point mutation, the A-->G3260 transition associated with maternally inherited myopathy and cardiomyopathy. To eliminate the possible influence of the autochthonous nuclear gene set, we fused myoblast-derived cytoplasts of a patient with a human tumoral cell line deprived of mtDNA (Rho degrees). The presence and amount of the mutant G3260 vs the wild-type A3260 were measured by solid phase minisequencing. We observed a marked reduction of the percentage of mutant mtDNA in the culture system compared with that measured in the donor's muscle biopsy, suggesting the presence of negative selection against the mutation. Furthermore, stable mitotic segregation of the two mtDNA populations was observed in 18 of 19 transformant clones, suggesting the presence of intraorganelle and possibly intracellular homoplasmy in the precursor cells of the donor. Several indexes of mtDNA-related respiratory capacity, including oxygen consumption, complex I- and complex IV-specific activities, and lactate production, were markedly abnormal in the clones containing a high proportion of mutant mtDNA, as compared with those containing homoplasmic wild-type mtDNA, possibly because of impaired mitochondrial protein synthesis. We conclude that (a) the A-->G3260 transition is indeed responsible for the mitochondrial disorder identified in the donor patient, and (b) transformant cybrid system gives direct evidence of the mitochondrial origin of a genetic disorder and should be adopted for the evaluation of the pathogenic potential of the mtDNA mutations.

Adult↗

PubMed1994

Remarkable recovery of visual function in a patient with Leber's optic neuropathy and multiple mutations of mitochondrial DNA.

Almost complete spontaneous recovery in visual function was observed in a male patient with Leber's hereditary optic neuropathy (LHON), in spite of the presence of several LHON-associated "major" and "minor" mutations of mitochondrial DNA. Our findings confirm that visual loss in LHON may be reversible, and challenge the hypothesis of a "synergistic" effect of multiple mtDNA mutations in the phenotypic expression of the disease.

Adolescent↗

PubMed1994

[Physical study of big fragments and search strategy of genes. Application to locus of infant spinal muscular atrophies].

Spinal muscular atrophies (SMA) represent the second most common fatal autosomal recessive disorder after cystic fibrosis. Childhood SMAs are divided into severe (type I) and mild forms (types II and III). By a combination of genetic and physical mapping, a YAC contig of the 5q13 region spanning the disease locus was constructed that showed the presence of low copy-repeats in this region. Allele segregation was analyzed at the closest genetic loci detected by markers C212 and C272 in 201 SMA families. Inherited and de novo deletions were observed in 10 SMA patients. Moreover, deletions were strongly suggested in at least 18% of SMA type I patients by the observation of marked heterozygosity deficiency for the loci studied. These results indicate that deletion events are statistically associated with the severe form of SMA.

Chromosome Mapping↗

PubMed1994

Mitochondrial diseases.

Mitochondrial diseases are heterogeneous and characterized by a primary defect of the mitochondrial energy output. Genetic defects of mitochondrial energy enzymes may be due to either nuclear DNA gene mutations or mitochondrial DNA (mtDNA) mutations. Among hereditary defects of nuclear-encoded mitochondrial enzymes, carnitine palmitoyltransferase II (CPT-II) deficiency and pyruvate dehydrogenase complex (PDHC) deficiency are of major interest to the neurologist. Several mutations in the CPT-II gene as well as in the X-linked E1 alpha subunit gene of PDHC have been reported and associated with different clinical phenotypes. mtDNA-related syndromes include mitochondrial encephalomyopathies (e.g. MELAS, MERRF, NARP, MIMyCa, etc.), 'pure' encephalopathies (e.g. LHON) and a few syndromes involving only non-neurological systems (e.g. Pearson's pancreas-bone marrow syndrome or diabetes mellitus). Three kinds of molecular lesions have been identified in mtDNA-related disorders: point mutations of protein-encoding mtDNA genes (mit- mutations), point mutations of mtDNA-tRNA genes (syn- mutations) and large-scale rearrangements of mtDNA (rho- mutations). Point mutations (mit- and syn+) are usually maternally inherited, while single large-scale mtDNA rearrangements are usually sporadic. Furthermore, mendelian traits leading to either qualitative or quantitative abnormalities of mtDNA (i.e. multiple mtDNA deletions and tissue-specific mtDNA depletion, respectively) are the first examples of genetic dysfunction of nuclear-mitochondrial communication. In most cases, the molecular detection of the known defects of mtDNA can be carried out by non-invasive techniques, thus making it an easy and relatively inexpensive procedure in the differential diagnosis of the mitochondrial disorders, a rapidly expanding area of clinical neurology.

DNA, Mitochondrial↗