Patrick Lestienne

Mitochondrial myopathies: divergences of genetic deletions, biochemical defects and the clinical syndromes

SummaryGenomic Southern analysis of muscle mitochondrial (mt) DNA from 16 patients with mitochondrial myopathies was performed; 14 of 16 patients had chronic progressive external ophthalmoplegia (CPEO), while 2 patients had mitochondrial myopathies without CPEO. Eleven patients with CPEO, including 5 who exhibited the complete triad of symptoms characteristic of the Kearns-Sayre syndrome (i.e. CPEO, retinal degeneration and heart block) had hetero-plasmic mtDNA with deletions ranging from 2.0 to 8.0 kb in length. There was no clear-cut correlation between the size and location of the deletions, on the one hand, and the histo-chemical and biochemical data or the severity of the disease, on the other.

Deletions of mitochondrial DNA in Kearns-Sayre syndrome and ocular myopathies: Genetic, biochemical and morphological studies

Genetic, biochemical and morphological investigations were conducted on skeletal muscle mitochondria from 6 cases of ocular myopathy: 4 cases with Kearns-Sayre syndrome (KSS) and 2 with chronic progressive external ophthalmoplegia. All of these 6 cases showed mitochondrial DNA (mtDNA) deletions in addition to normal sized DNA in the quadriceps muscle. The deletions ranging from 3 to 8 kbp were also mapped between nucleotides 5500 and 16000 by Southern blot. The deleted genes encoded for some subunits of complexes I, IV, V and 5-10 tRNAS. The boundaries of the deletions have been sequenced in three patients. Five patients had mitochondrial respiratory chain deficiency in complex I as shown by the low oxygen consumption in isolated mitochondria using three NAD(+)-linked substrates. Mitochondria with an abnormal ultrastructure were also observed in 2 cases. A good relationship between the cytochrome c oxidase deficiency and the amount of deleted mtDNA was shown in our present investigations.

Mitochondrial DNA in Postmortem Brain from Patients with Parkinson's Disease

To the Editors: Using Southern blot analysis with mitochondrial DNA (mtDNA) as a probe, we reported that the mtDNA was normal in the putamen, the cortex, and the substantia nigra of patients with Parkinsons disease, where a complex I defect had been observed (Lestienne et al., 1990). However, Ikebe et al. (1 990) using the much more sensitive technique of polymerase chain reaction (PCR) (Saiki et al., 1985) recently detected small amounts of the mtDNA deleted genome in the striatum of patients with Parkinsons disease and senescence. We have repeated this experiment and found that in addition to numerous bands, the deleted genome was present in a very low amount even in a control. This is shown in Fig. 1, where we have done a PCR shifi assay using two different forward and reverse oligonucleotide primers to amplify the

Mitochondrial DNA mutations in human diseases: a review

Human mitochondrial diseases have been associated recently with mitochondrial DNA mutations, duplications and deletions which impair the protein synthesis of the mitochondrial subunits of the respiratory chain complexes. A constant feature is the coincident presence of the mutated and wild type genomes which provide heteroplasmy. The clinical expression of these diseases depends on the relative expression of each kind of mitochondrial DNA in the various tissues, which in turn affects the production of ATP in these tissues. Research on nuclear gene products interfering with mtDNA or with its gene products is the next step towards understanding the etiology of these diseases.

Different copy numbers of apparently identically deleted mitochondrial DNA in tissues from a patient with Kearns-Sayre syndrome detected by PCR.

An apparently identical deletion of 4.977 bp in length (position 8,483-13,459) was detectable in the mitochondrial DNA from skeletal muscle, heart muscle, kidney, and liver of a patient with Kearns-Sayre syndrome. The proportion of deleted genome varied from 60% for the skeletal muscle to 15% for heart muscle and kidney, and was below 5% in the liver. The mtDNA heteroplasmy of the liver was only detectable after amplification by PCR. In skeletal and heart muscle histochemical and immunocytochemical findings concerning cytochrome c oxidase were in good correlation with the proportion of deleted mitochondrial DNA.

Steady state levels of mitochondrial and nuclear oxidative phosphorylation transcripts in Kearns-Sayre syndrome

The steady state levels of both mitochondrial and nuclear transcripts were examined in a Kearns-Sayre syndrome patient harboring a heteroplasmic 7.7 kb mitochondrial DNA deletion. Transcripts originating from the genes located outside of the deletion were present in similar amounts to those of control samples, with the transcript levels of each tissue linked to its oxidative phosphorylation capacities. Transcripts originating from genes within the deletion were reduced according to the percentage of mtDNA deleted molecules in the tissue. The fusion transcript resulting from the rearranged genome is expressed in all the tissues tested and its level is related to the amount of the deleted mtDNA. The RNA levels from three nuclear genes encoding two of the Adenine Nucleotide Translocator isoforms (ANT1 and 2) and the beta subunit of the ATPsynthase (ATPsyn beta) were significantly induced in the different tissues independently of the percentage of deleted mtDNA molecules. In contrast, the ANT1 and ATPsyn beta levels were decreased in skeletal muscle. This result could be related to the different distribution of the deleted molecules in tissues.

Base composition at mtDNA boundaries suggests a DNA triple helix model for human mitochondrial DNA large-scale rearrangements.

Different mechanisms have been proposed to account for mitochondrial DNA (mtDNA) instability based on the presence of short homologous sequences (direct repeats, DR) at the potential boundaries of mtDNA rearrangements. Among them, slippage-mispairing of the replication complex during the asymmetric replication cycle of the mammalian mitochondrial DNA has been proposed to account for the preferential localization of deletions. This mechanism involves a transfer of the replication complex from the first neo-synthesized heavy (H) strand of the DR1, to the DR2, thus bypassing the intervening sequence and producing a deleted molecule. Nevertheless, the nature of the bonds between the DNA strands remains unknown as the forward sequence of DR2, beyond the replication complex, stays double-stranded. Here, we have analyzed the base composition of the DR at the boundaries of mtDNA deletions and duplications and found a skewed pyrimidine content of about 75% in the light-strand DNA template. This suggests the possible building of a DNA triple helix between the G-rich neo-synthesized DR1 and the base-paired homologous G.C-rich DR2. In vitro experiments with the purified human DNA polymerase gamma subunits enabled us to show that the third DNA strand may be used as a primer for DNA replication, using a template with the direct repeat forming a hairpin, with which the primer could initiate DNA replication. These data suggest a novel molecular basis for mitochondrial DNA rearrangements through the distributive nature of the DNA polymerase gamma, at the level of the direct repeats. A general model accounting for large-scale mitochondrial DNA deletion and duplication is proposed. These experiments extend to a DNA polymerase from an eucaryote source the use of a DNA triple helix strand as a primer, like other DNA polymerases from phage and bacterial origins.

Evidence for a direct role of the DNA polymerase gamma in the replication of the human mitochondrial DNA in vitro.

Indirect experiments suggest that DNA polymerase gamma is involved in the mitochondrial DNA replication process. This report describes an in vitro mitochondrial DNA replication assay directed by the origin of replication of the Heavy strand mt DNA. The assay requires all four dNTP, rNTP and an ATP regenerating system. Nuclease digestion experiments show that specific events occur at the mt origin of replication. Antibodies raised against the purified DNA polymerase gamma inhibit the DNA replication reaction.

Immunocytological and histochemical correlation in Kearns-Sayre syndrome with mtDNA deletion and partial cytochrome c oxidase deficiency in skeletal muscle

We report histochemical, immunocytochemical, biochemical and molecular studies of skeletal muscle from a 23-year-old man with Kearns-Sayre syndrome. Southern blot analysis revealed a 4.7 kb heteroplasmic deletion of the mitochondrial DNA mapping within genes coding for subunits of complexes I, IV and V of the respiratory chain and for tRNA. Cytochrome c oxidase activity was decreased by 30% in isolated muscle mitochondria, without alteration of the Km. Histochemical and immunocytochemical correlation studies for cytochrome c oxidase revealed a lack of activity in 34% of individual muscle fibers including all the typical ragged-red fibers and a low percentage of immunodeficient fibers.

Uneven distribution of mitochondrial DNA mutation in MERRF dizygotic twins

A new family of myoclonic epilepsy with ragged-red fibers (MERRF) was studied at clinical, histological, biochemical and molecular genetic levels. There was a remarkable variation in the age of onset, the clinical presentation and the severity of symptoms. Multiple defects affecting respiratory chain complexes I, III and IV were detected in 2 patients. The point mutation at 8344 of the mitochondrial genome was found in all the maternal lineage with a relatively narrow range of variation in the percentage of mutant mitochondrial genomes. The one exception was represented by a set of dizygotic twins, one clinically affected and showing high proportions of mutant mitochondrial DNAs (mtDNAs) in blood cells, while the other was asymptomatic and showed very small amounts of mutant mt-DNAs in blood and skin. This could suggest an early segregation of the mitochondrial genome during ovogenesis.