A Tunisian patient with Pearson syndrome harboring the 4.977kb common deletion associated to two novel large-scale mitochondrial deletions

Pearson syndrome (PS) is a multisystem disease including refractory anemia, vacuolization of marrow precursors and pancreatic fibrosis. The disease starts during infancy and affects various tissues and organs, and most affected children die before the age of 3 years. Pearson syndrome is caused by de novo large-scale deletions or, more rarely, duplications in the mitochondrial genome. In the present report, we described a Pearson syndrome patient harboring multiple mitochondrial deletions which is, in our knowledge, the first case described and studied in Tunisia. In fact, we reported the common 4.977 kb deletion and two novel heteroplasmic deletions (5.030 and 5.234 kb) of the mtDNA. These deletions affect several protein-coding and tRNAs genes and could strongly lead to defects in mitochondrial polypeptides synthesis, and impair oxidative phosphorylation and energy metabolism in the respiratory chain in the studied patient.     

Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalomyopathy and cardiomyopathy

The heart mitochondria isolated from a patient with hypertrophic cardiomyopathy associated with mitochondrial encephalomyopathy were analyzed by immunoblotting using specific antibody against each of the purified mitochondrial energy transducing complexes from beef heart. Subunits of NADH-ubiquinone oxidoreductase (Complex I) were markedly decreased and those of cytochrome c oxidase (Complex IV) were decreased to some extent, but the deficiency of any of these subunits was only partial. On the other hand, the contents of subunits of ubiquinol-cytochrome c oxidoreductase (Complex III) were normal. These results suggest that the decreased levels of some of the Complex I subunits might be the primary cause of disorder in this patient.     

Site-specific deletions of the mitochondrial genome in the Pearson marrow-pancreas syndrome.

The Pearson marrow-pancreas syndrome is a fatal disorder involving the hematopoietic system and the exocrine pancreas in early infancy. We have previously shown that this disease results from a widespread defect of oxidative phosphorylation. Here, we describe deletions of the mitochondrial (mt) genome between repeated 8- to 13-bp sequences as consistent features of the disease. Studying a series of nine unrelated children, including the patient originally reported by H. Pearson, we found five different types of direct repeats at the boundaries of the mtDNA deletions and we provided evidence for conservation of the 3'-repeated sequence in the deletions. In addition, we found a certain degree of homology between the nucleotide composition of the direct repeats and several structures normally involved in mtDNA replication and mtRNA processing. These results are consistent either with the recognition and cleavage of a particular DNA sequence with a factor of still unknown origin or with a homologous recombination between direct-repeat mtDNA sequences in the Pearson syndrome.     

Autosomal recessive Wolfram syndrome associated with an 8.5-kb mtDNA single deletion.

Wolfram syndrome (MIM 222300) is characterized by optic atrophy, diabetes mellitus, diabetes insipidus, neurosensory hearing loss, urinary tract abnormalities, and neurological dysfunction. The association of clinical manifestations in tissues and organs unrelated functionally or embryologically suggested the possibility of a mitochondrial implication in the disease, which has been demonstrated in two sporadic cases. Nonetheless, familial studies suggested an autosomal recessive mode of transmission, and recent data demonstrated linkage with markers on the short arm of human chromosome 4. The patient reported here, as well as her parents and unaffected sister, carried a heteroplasmic 8.5-kb deletion in mtDNA. The deletion accounted for 23% of mitochondrial genomes in lymphocytes from the patient and approximately 5% in the tissues studied from members of her family. The presence of the deletion in the patient in a proportion higher than in her unaffected parents suggests a putative defect in a nuclear gene that acts at the mitochondrial level.     

Variation in the levels of complex I subunits among tissues in a patient with mitochondrial encephalomyopathy and renal dysfunction

Enzymic activity and the levels of immunochemically detectable subunits of NADH-ubiquinone oxidoreductase (Complex I) were measured in the mitochondria from various tissues of a patient with mitochondrial encephalomyopathy and renal dysfunction. Rotenone-sensitive NADH-cytochrome c reductase activity was decreased in all the tissues examined, but the degree of deficiency varied from tissue to tissue. The levels of subunits in Complex I were decreased roughly in parallel with the activity in each tissue. These results indicate that the apparently tissue-specific manifestation of symptoms depends mainly on the levels of subunits in Complex I.     

Coexistence of mitochondrial and nuclear DNA mutations in a woman with mitochondrial encephalomyopathy and double cortex

We describe a 16-year-old girl with mental retardation, myoclonic epilepsy, ataxia, mitochondrial myopathy, sensorineural hearing loss, lactic acidosis, and MRI evidence of diffuse subcortical laminar heterotopia and agyria/pachygyria. Restriction fragment length polymorphism (RFLP) and DNA sequence analyses revealed two pathogenic mutations: a heteroplasmic m.3243A > G in muscle and blood, and a new heterozygous insertion at nt697 in the doublecortin gene (DCX), resulting in a frameshift after amino acid residue 232, with a premature stop codon at amino acid residue 244. This is yet another example of genetic “double trouble” resulting in a complex phenotype.     

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 enxyme 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.     

Compound mutations of PEO1 and TYMP in a progressive external ophthalmoplegia patient with incomplete mitochondrial neurogastrointestinal encephalomyopathy phenotype

The Mendelian inherited progressive external ophthalmoplegia (PEO) and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) are genetically heterogeneous mitochondrial diseases caused by nuclear-mitochondrial intergenomic defects. The PEO1 and TYMP nuclear genes are closely related in the machinery of the mitochondrial DNA (mtDNA) replication. Mutations in PEO1 and TYMP genes usually cause autosomal dominant PEO, and autosomal recessive MNGIE. We identified a PEO family of Korean origin with additional phenotype of incomplete MNGIE symptom (Family ID: MT16). The entire mitochondrial genome and all coding exons of PEO1, TYMP, ANT1, POLG1, POLG2, DGUOK, and TK2 nuclear genes were sequenced. Clinical information was obtained through history taking, physical examinations, clinical observations, and electrophysiological investigations. Muscle biopsy of left biceps brachii and shoulder magnetic resonance imaging (MRI) were undertaken. We found two heterozygous mutations, Arg374Gln in PEO1 and Glu106Gln in TYMP from the proband who showed complex phenotypes of a typical PEO and late-onset incomplete MNGIE. The PEO1 Arg374Gln has been reported in several PEO patients, but TYMP Glu106Gln has not been reported. Neither large deletion nor causative point mutations were observed in the mtDNA. We suggest that the heterozygous TYMP mutation might affect complex phenotypes as a secondary genetic cause in the co-presence of PEO1 mutation.     

Intragenic inversion of mtDNA: a new type of pathogenic mutation in a patient with mitochondrial myopathy

We report an unusual molecular defect in the mitochondrially encoded ND1 subunit of NADH ubiquinone oxidoreductase (complex I) in a patient with mitochondrial myopathy and isolated complex I deficiency. The mutation is an inversion of seven nucleotides within the ND1 gene, which maintains the reading frame. The inversion, which alters three highly conserved amino acids in the polypeptide, was heteroplasmic in the patient's muscle but was not detectable in blood. This is the first report of a pathogenic inversion mutation in human mtDNA.     

Nuclear complementation restores mtDNA levels in cultured cells from a patient with mtDNA depletion.

We have studied cultured skin fibroblasts from a patient with a fatal mitochondrial disease manifesting soon after birth. These fibroblasts were found to grow only in the presence of pyruvate and uridine, a characteristic of cells lacking mtDNA (rho0 cells). Southern blot and PCR analyses confirmed that the patient's fibroblasts contained less than 2% of control levels of mtDNA. Biochemical analyses indicated that the activities of all the respiratory-chain enzymes were severely decreased in mitochondria isolated from these fibroblasts. In order to elucidate the underlying molecular defect, cell fusions were performed between enucleated fibroblasts from this patient and a human-derived rho0 cell line (rho0 A549.B2). The resulting cybrids were plated in medium lacking pyruvate and uridine, to select for the restoration of respiratory-chain function. Complementation was observed between the nuclear genome of the rho0 A549.B2 cells and the mtDNA of the patient's cells, restoring mtDNA levels and respiratory-chain function in the cybrid cells. These results indicate that mtDNA depletion in our patient is under the control of the nuclear genome.     

Mice with mitochondrial complex I deficiency develop a fatal encephalomyopathy

To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until approximately 5 weeks of age, when ataxic signs began, progressing to death at approximately 7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters.     

The mutation rate of the human mtDNA deletion mtDNA4977.

The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.     

Identical mitochondrial DNA deletion in a woman with ocular myopathy and in her son with pearson syndrome

Single deletions of mitochondrial DNA (mtDNA) are associated with three major clinical conditions: Kearns-Sayre syndrome, a multisystem disorder; Pearson syndrome (PS), a disorder of the hematopoietic system; and progressive external ophthalmoplegia (PEO), primarily affecting the ocular muscles. Typically, single mtDNA deletions are sporadic events, since the mothers, siblings, and offspring of affected individuals are unaffected. We studied a woman who presented with PEO, ptosis, and weakness of pharyngeal, facial, neck, and limb muscles. She had two unaffected children, but another of her children, an infant son, had sideroblastic anemia, was diagnosed with PS, and died at age 1 year. Morphological analysis of a muscle biopsy sample from the mother showed cytochrome c oxidase-negative ragged-red fibers-a typical pattern in patients with mtDNA deletions. Southern blot analysis using multiple restriction endonucleases and probed with multiple mtDNA fragments showed that both the mother and her infant son harbored an identical 5,355-bp single deletion in mtDNA, without flanking direct repeats. The deletion was the only abnormal species of mtDNA identified in both patients, and there was no evidence for duplications. We conclude that, although the vast majority of single large-scale deletions in mtDNA are sporadic, in rare cases, single deletions can be transmitted through the germline.     

Autosomal dominant deletions of the mitochondrial genome in a case of progressive encephalomyopathy.

Multiple deletions of the mitochondrial genome were found in a family in which the proband had ataxia and ketoacidotic comas. A progressive multiorgan involvement appeared in the course of the disease, and histopathological investigation demonstrated mitochondrial myopathy features with ragged red fibers. A defect of oxidative phosphorylation was found in both skeletal muscle and lymphocytes. It is surprising that various mtDNA deletions were detected both in the proband and in his healthy mother and maternal aunt but not in the rest of the maternal progeny. All the deletions were located between Cox II and cytochrome b genes, and short (4-5 bp) repeated sequences were consistently present at the boundaries of the rearrangements in different tissues. Therefore, the deletions appear not to be transmitted per se but to be inherited in a Mendelian manner, being possibly dominant. Both the Mendelian inheritance of the trait and the variety of the deletions in carriers suggest that a nuclearly encoded factor(s) might be involved in the triggering of the deletions. However, the presence of the rearrangements in healthy individuals raises the question of whether mtDNA deletions actually cause the clinical expression of the disease.     

Maternal origin of a de novo chromosome 8 deletion in a patient with Langer-Giedion syndrome

Summary The anonymous DNA probe L32, which defines the D8S48 locus within the Langer-Giedion syndrome chromosome region on the long arm of chromosome 8, was used to search for a common restriction fragment length polymorphism. A HindIII and an MspI polymorphism were detected (polymorphism information contents 0.25 and 0.19, respectively). Both polymorphisms were informative in the family of a Langer-Giedion patient carrying a de novo interstitial deletion 8q23-24.1. Lack of transmission of a maternal haplotype indicates that this deletion occurred during maternal gametogenesis. This finding contrasts with the frequent paternal origin of mutations in other microdeletion syndromes.     

Germ-line deletions of mtDNA in mitochondrial myopathy.

mtDNA encodes subunits of the electron transport chain and is exclusively maternally inherited in mammals. It has been suggested that mtDNA might be the site of some of the mutations causing a group of human disorders called the "mitochondrial myopathies," because these may both be (1) accompanied by defects in the electron transport chain and (2) display a maternal pattern of inheritance. However, all of the deletions and duplications of mtDNA which occur in these patients have been sporadic, apart from families in whom affected members all carry different deletions suggesting a mutant autosomal dominantly inherited nuclear gene with de novo deletions in each individual. We present the first evidence for the presence of deleted mtDNAs in the germ line in these disorders. The patient carries a higher level of deleted mtDNAs than do his relatives, corresponding to severity of symptoms and consistent with a predicted dosage effect. "Selfishness" of deleted mtDNAs is probably one of the factors over and above random segregation of a small number of "founder" mtDNAs (the bottleneck hypothesis) which may be invoked to explain the usual distribution of mtDNAs in different tissues of patients with mtDNA deletions.