Pascal Reynier

Multi-system neurological disease is common in patients with OPA1 mutations

Additional neurological features have recently been described in seven families transmitting pathogenic mutations in OPA1, the most common cause of autosomal dominant optic atrophy. However, the frequency of these syndromal ‘dominant optic atrophy plus’ variants and the extent of neurological involvement have not been established. In this large multi-centre study of 104 patients from 45 independent families, including 60 new cases, we show that extra-ocular neurological complications are common in OPA1 disease, and affect up to 20% of all mutational carriers. Bilateral sensorineural deafness beginning in late childhood and early adulthood was a prominent manifestation, followed by a combination of ataxia, myopathy, peripheral neuropathy and progressive external ophthalmoplegia from the third decade of life onwards. We also identified novel clinical presentations with spastic paraparesis mimicking hereditary spastic paraplegia, and a multiple sclerosis-like illness. In contrast to initial reports, multi-system neurological disease was associated with all mutational subtypes, although there was an increased risk with missense mutations [odds ratio = 3.06, 95% confidence interval = 1.44–6.49; P = 0.0027], and mutations located within the guanosine triphosphate-ase region (odds ratio = 2.29, 95% confidence interval = 1.08–4.82; P = 0.0271). Histochemical and molecular characterization of skeletal muscle biopsies revealed the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in the majority of patients harbouring OPA1 mutations, even in those with isolated optic nerve involvement. However, the cytochrome c oxidase-deficient load was over four times higher in the dominant optic atrophy + group compared to the pure optic neuropathy group, implicating a causal role for these secondary mitochondrial DNA defects in disease pathophysiology. Individuals with dominant optic atrophy plus phenotypes also had significantly worse visual outcomes, and careful surveillance is therefore mandatory to optimize the detection and management of neurological disability in a group of patients who already have significant visual impairment.

OPA1 R445H mutation in optic atrophy associated with sensorineural deafness

The heterozygous R445H mutation in OPA1 was found in five patients with optic atrophy and deafness. Audiometry suggested that the sensorineural deafness resulted from auditory neuropathy. Skin fibroblasts showed hyperfragmentation of the mitochondrial network, decreased mitochondrial membrane potential, and adenosine triphosphate synthesis defect. In addition, OPA1 was found to be widely expressed in the sensory and neural cochlear cells of the guinea pig. Thus, optic atrophy and deafness may be related to energy defects due to a fragmented mitochondrial network. Ann Neurol 2005

Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors

BackgroundRecent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far.MethodsWe used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1), and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1), and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA).ResultsUnlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage.ConclusionOur results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis.

Impact of various handling and storage conditions on quantitative detection of hepatitis C virus RNA

BACKGROUND/AIMS Both HCV RNA viral load and HCV genotype have been described as important predicting factors determining the response to interferon in chronic hepatitis C. To investigate whether processing and storage conditions might influence the stability and could alter the concentration of the HCV RNA in serum, quantification of HCV RNA was performed by branched DNA assay. METHODS We studied serum samples obtained from seven patients with histologically proven chronic hepatitis C. These were subjected to the following physical conditions: (1) immediate quantification, (2) storage at room temperature for 5 days, (3) storage at 4 degrees C for 5 days, (4) storage at -20 degrees C for 5 days, (5) storage at -80 degrees C for 5 days, (6) five freeze-thaw cycles, (7) blood unspun for 4 h at room temperature then centrifuged and stored at -80 degrees C for 5 days, (8) storage at 4 degrees C for 6 months, (9) storage at -20 degrees C for 6 months, (10) storage at -80 degrees C for 6 months. RESULTS A loss of 100% HCV RNA titers was observed after storage at RT for 5 days and then storage at 4 degrees C for 6 months. A surprising decrease of HCV RNA titer (15.6%) was observed in sera stored for 5 days at -20 degrees C. Five freeze-thaw cycles resulted in a 16% decrease of the HCV RNA level. When centrifugation was performed after a 4 h delay at room temperature, a significant loss of HCV RNA titers of 29.5% was observed. Long-term stability (6 months) was observed at -80 degrees C with a slight loss of about of 10% HCV RNA titers, but a significant decrease in HCV RNA of 23% was observed at -20 degrees C. The reproducibility of the bDNA assay on five patient samples was performed eight times in duplicate and showed an average coefficient of variation of 9.1%. CONCLUSIONS These data confirm the importance of storage and handling in measuring the amount of HCV RNA in clinical samples.

OPA3 gene mutations responsible for autosomal dominant optic atrophy and cataract

Hereditary optic atrophy is a generic term that refers to a heterogeneous group of genetic disorders for which several modes of inheritance have been described.1 The most common forms of optic atrophy are autosomal dominant optic atrophy (ADOA, OMIM 165500) and Leber’s hereditary optic neuropathy (LHON, OMIM 53500). ADOA, which generally starts in childhood, is characterised by a progressive decrease in visual acuity, blue-yellow dyschromatopsia, loss of sensitivity in the central visual field, and optic nerve pallor. Mutations in the optic atrophy 1 ( OPA1 ) gene, located on chromosome 3q28–q29, are implicated in about 60–80% of the cases of ADOA.1–4 OPA1 encodes for a mitochondrial dynamin related protein. This protein, anchored to the mitochondrial inner membrane, contributes to mitochondrial structure and biogenesis.5,6 A second gene involved in ADOA, not yet identified, has been mapped to chromosome 18q ( OPA4 , OMIM 605293).7 LHON, which is caused by specific mutations in mitochondrial DNA, is inherited maternally.8 It is characterised by severe bilateral optic atrophy responsible for acute or subacute visual loss, usually starting between the ages of 18 and 35. Other forms of hereditary optic atrophy include X linked optic atrophy (XLAO, OPA2 , OMIM 311050)9 and autosomal recessive optic atrophy (AROA), for which a first locus has recently been mapped to chromosome 8q.10 Finally, more than 15 disorders—mostly inherited in the autosomal recessive mode—have combined optic atrophy and extraocular anomalies. Among these syndromic optic atrophies, type III 3-methylglutaconic aciduria (MGA) (OMIM 258501), also known as the Costeff syndrome11 or the optic atrophy plus syndrome, consists of early onset bilateral optic atrophy, later onset spasticity, extrapyramidal signs, and cognitive deficit. Urinary excretion of 3-methyl glutaconic acid and increased plasma 3-methylglutaric acid levels are the hallmarks of MGA.12 Linkage analyses, undertaken in MGA …

EFFECTS OF OPA1 MUTATIONS ON MITOCHONDRIAL MORPHOLOGY AND APOPTOSIS: RELEVANCE TO ADOA PATHOGENESIS *

To characterize the molecular links between type‐1 autosomal dominant optic atrophy (ADOA) and OPA1 dysfunctions, the effects of pathogenic alleles of this dynamin on mitochondrial morphology and apoptosis were analyzed, either in fibroblasts from affected individuals, or in HeLa cells transfected with similar mutants. The alleles were missense substitutions in the GTPase domain (OPA1G300E and OPA1R290Q) or deletion of the GTPase effector domain (OPA1Δ58). Fragmentation of mitochondria and apoptosis increased in OPA1R290Q fibroblasts and in OPA1G300E transfected HeLa cells. OPA1Δ58 did not influence mitochondrial morphology, but increased the sensitivity to staurosporine of fibroblasts. In these cells, the amount of OPA1 protein was half of that in control fibroblasts. We conclude that GTPase mutants exert a dominant negative effect by competing with wild‐type alleles to integrate into fusion‐competent complexes, whereas C‐terminal truncated alleles act by haplo‐insufficiency. We present a model where antagonistic fusion and fission forces maintain the mitochondrial network, within morphological limits that are compatible with cellular functions. In the retinal ganglion cells (RGCs) of patients suffering from type‐1 ADOA, OPA1‐driven fusion cannot adequately oppose fission, thereby rendering them more sensitive to apoptotic stimuli and eventually leading to optic nerve degeneration. J. Cell. Physiol. 211: 423–430, 2007.

OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution

Eukaryotic cells harbor a small multiploid mitochondrial genome, organized in nucleoids spread within the mitochondrial network. Maintenance and distribution of mitochondrial DNA (mtDNA) are essential for energy metabolism, mitochondrial lineage in primordial germ cells, and to prevent mtDNA instability, which leads to many debilitating human diseases. Mounting evidence suggests that the actors of the mitochondrial network dynamics, among which is the intramitochondrial dynamin OPA1, might be involved in these processes. Here, using siRNAs specific to OPA1 alternate spliced exons, we evidenced that silencing of the OPA1 variants including exon 4b leads to mtDNA depletion, secondary to inhibition of mtDNA replication, and to marked alteration of mtDNA distribution in nucleoid and nucleoid distribution throughout the mitochondrial network. We demonstrate that a small hydrophobic 10-kDa peptide generated by cleavage of the OPA1-exon4b isoform is responsible for this process and show that this peptide is embedded in the inner membrane and colocalizes and coimmunoprecipitates with nucleoid components. We propose a novel synthetic model in which a peptide, including two trans-membrane domains derived from the N terminus of the OPA1-exon4b isoform in vertebrates or from its ortholog in lower eukaryotes, might contribute to nucleoid attachment to the inner mitochondrial membrane and promotes mtDNA replication and distribution. Thus, this study places OPA1 as a direct actor in the maintenance of mitochondrial genome integrity.

Mitochondrial DNA in the oocyte and the developing embryo.

Mitochondria play a primary role in cellular energetic metabolism, homeostasis, and death. They possess their own multicopy genome, which is maternally transmitted. Mitochondria are directly involved at several levels in the reproductive process since their functional status influences the quality of oocytes and contributes to the process of fertilization and embryonic development. This chapter discusses recent findings concerning mitochondrial DNA content and its expression during oogenesis, fertilization, and early embryonic development.

Hereditary optic neuropathies share a common mitochondrial coupling defect

Hereditary optic neuropathies are heterogeneous diseases characterized by the degeneration of retinal ganglion cells leading to optic nerve atrophy and impairment of central vision. We found a common coupling defect of oxidative phosphorylation in fibroblasts of patients affected by autosomal dominant optic atrophy (mutations of OPA1), autosomal dominant optic atrophy associated with cataract (mutations of OPA3), and Lebers hereditary optic neuropathy, a disorder associated with point mutations of mitochondrial DNA complex I genes. Interestingly, the energetic defect was significantly more pronounced in Lebers hereditary optic neuropathy and autosomal dominant optic atrophy patients with a more complex phenotype, the so‐called plus phenotype. Ann Neurol 2008

Molecular screening of 980 cases of suspected hereditary optic neuropathy with a report on 77 novel OPA1 mutations.

We report the results of molecular screening in 980 patients carried out as part of their work‐up for suspected hereditary optic neuropathies. All the patients were investigated for Lebers hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (ADOA), by searching for the ten primary LHON‐causing mtDNA mutations and examining the entire coding sequences of the OPA1 and OPA3 genes, the two genes currently identified in ADOA. Molecular defects were identified in 440 patients (45% of screened patients). Among these, 295 patients (67%) had an OPA1 mutation, 131 patients (30%) had an mtDNA mutation, and 14 patients (3%), belonging to three unrelated families, had an OPA3 mutation. Interestingly, OPA1 mutations were found in 157 (40%) of the 392 apparently sporadic cases of optic atrophy. The eOPA1 locus‐specific database now contains a total of 204 OPA1 mutations, including 77 novel OPA1 mutations reported here. The statistical analysis of this large set of mutations has led us to propose a diagnostic strategy that should help with the molecular work‐up of optic neuropathies. Our results highlight the importance of investigating LHON‐causing mtDNA mutations as well as OPA1 and OPA3 mutations in cases of suspected hereditary optic neuropathy, even in absence of a family history of the disease.