Julien Cassereau

Mitochondrial complex I deficiency in GDAP1-related autosomal dominant Charcot-Marie-Tooth disease (CMT2K)

Mutations in GDAP1, an outer mitochondrial membrane protein responsible for recessive Charcot-Marie-Tooth disease (CMT4A), have also been associated with CMT2K, a dominant form of the disease. The three CMT2K patients we studied carried a novel dominant GDAP1 mutation, C240Y (c.719G > A). Mitochondrial respiratory chain complex I activity in fibroblasts from CMT2K patients was 40% lower than in controls, whereas the tubular mitochondria were 33% larger in diameter and the mitochondrial mass was 20% greater. Thus, besides the regulatory role GDAP1 plays in mitochondrial network dynamics, it may also be involved in energy production and in the control of mitochondrial volume.

Hereditary spastic paraplegia-like disorder due to a mitochondrial ATP6 gene point mutation.

Hereditary spastic paraplegia refers to a genetically heterogeneous syndrome. We identified five members of a family suffering from a late-onset spastic paraplegia-like disorder, carrying the homoplasmic m.9176 T>C mutation in the mitochondrial ATP6 gene. The clinical severity of the disease observed in the family was correlated with the biochemical and assembly defects of the ATP synthase. The m.9176 T>C mutation has been previously associated to Leigh syndrome or familial bilateral striatal necrosis. Other factors such as modifying genes may be involved in the phenotypic expression of the disease. The family belongs to the mitochondrial haplogroup J, previously shown to play a role in modulating the phenotype of mitochondrial diseases and be associated with longevity. Moreover other nuclear modifying genes or environmental factors may contribute to the disease phenotype. This finding extends the genetic heterogeneity of the hereditary spastic paraplegia together with the clinical spectrum of mutations of the ATP6 gene.

Mitochondrial dysfunction and pathophysiology of Charcot–Marie–Tooth disease involving GDAP1 mutations

Charcot-Marie-Tooth (CMT) disease represents a large group of clinically and genetically heterogeneous disorders leading to inherited peripheral neuropathies affecting motor and sensory neurons. Mutations in the ganglioside-induced differentiation-associated-protein 1 gene (GDAP1), which encodes a protein anchored to the mitochondrial outer membrane, are usually associated with the recessive forms of CMT disease and only rarely with the autosomal dominant forms. The function of GDAP1 is not fully understood but it plays a role in mitochondrial dynamics by promoting fission events. We present an overview of GDAP1 and the corresponding protein together with the complete spectrum of the 41 gene mutations described so far. We examine the relationship between the genotype and the phenotype in the various forms of CMT disease related to GDAP1 mutations, and discuss the pathophysiological hypotheses that link peripheral neuropathies to mitochondrial dysfunction and GDAP1 mutations. The meta-analysis of the literature reveals the great heterogeneity of phenotypic presentations and shows that the recessive forms of CMT disease, i.e. CMT4A and AR-CMT2, are far more severe than the dominant form, i.e. CMT2K. Among patients with recessive forms of the disease, those carrying truncating mutations are more seriously affected, often becoming wheelchair-bound before the end of the third decade. At the neuronal level, GDAP1 mutations may lead to perturbed axonal transport and impaired energy production as in other neurodegenerative diseases due to mutations in genes involved in mitochondrial dynamics.

Phenotypic spectrum of MFN2 mutations in the Spanish population

Introduction The most common form of axonal Charcot–Marie–Tooth (CMT) disease is type 2A, caused by mutations in the mitochondrial GTPase mitofusin 2 (MFN2). Objective The objective of our study is to establish the incidence of MFN2 mutations in a cohort of Spanish patients with axonal CMT neuropathy. Material and Methods Eighty-five families with suspected axonal CMT were studied. All MFN2 exons were studied through direct sequencing. A bioenergetics study in fibroblasts was conducted using a skin biopsy taken from a patient with an Arg468His mutation. Results Twenty-four patients from 14 different families were identified with nine different MFN2 mutations (Arg94Trp, Arg94Gln, Ile203Met, Asn252Lys, Gln276His, Gly296Arg, Met376Val, Arg364Gln and Arg468His). All mutations were found in the heterozygous state and four of these mutations had not been described previously. MFN2 mutations were responsible for CMT2 in 16% ± 7% of the families studied and in 30.8 ± 14.2% (12/39) of families with known dominant inheritance. The bioenergetic studies in fibroblasts show typical results of MFN2 patients with a mitochondrial coupling defect (ATP/O) and an increase of the respiration rate linked to complex II. Conclusion It is concluded that mutations in MFN2 are the most frequent cause of CMT2 in this region. The Arg468His mutation was the most prevalent (6/14 families), and our study confirms that it is pathological, presenting as a neuropathy in a mild to moderate degree. This study also demonstrates the value of MFN2 studies in cases of congenital axonal neuropathy, especially in cases of dominant inheritance, severe clinical symptoms or additional symptoms such as optic atrophy.

Ethambutol-induced optic neuropathy linked to OPA1 mutation and mitochondrial toxicity.

Ethambutol (EMB), widely used in the treatment of tuberculosis, has been reported to cause Lebers hereditary optic neuropathy in patients carrying mitochondrial DNA mutations. We study the effect of EMB on mitochondrial metabolism in fibroblasts from controls and from a man carrying an OPA1 mutation, in whom the drug induced the development of autosomal dominant optic atrophy (ADOA). EMB produced a mitochondrial coupling defect together with a 25% reduction in complex IV activity. EMB induced the formation of vacuoles associated with decreased mitochondrial membrane potential and increased fragmentation of the mitochondrial network. Mitochondrial genetic variations may therefore be predisposing factors in EMB-induced ocular injury.

Inherited peripheral neuropathies due to mitochondrial disorders

Mitochondrial disorders (MIDs) are frequently responsible for neuropathies with variable severity. Mitochondrial diseases causing peripheral neuropathies (PNP) may be due to mutations of mitochondrial DNA (mtDNA), as is the case in MERRF and MELAS syndromes, or to mutations of nuclear genes. Secondary abnormalities of mtDNA (such as multiple deletions of muscle mtDNA) may result from mitochondrial disorders due to mutations in nuclear genes involved in mtDNA maintenance. This is the case in several syndromes caused by impaired mtDNA maintenance, such as Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO) due to recessive mutations in the POLG gene, which encodes the catalytic subunit of mtDNA polymerase (DNA polymerase gamma), or Mitochondrial Neuro-Gastro-Intestinal Encephalomyopathy (MNGIE), due to recessive mutations in the TYMP gene, which encodes thymidine phosphorylase. The last years have seen a growing list of evidence demonstrating that mitochondrial bioenergetics and dynamics might be dysfunctional in axonal Charcot-Marie-Tooth disease (CMT2), and these mechanisms might present a common link between dissimilar CMT2-causing genes.

Relations génotype-phénotype dans la maladie de Charcot-Marie-Tooth associée aux mutations du gène GDAP1

Introduction Le gene GDAP1 (Ganglioside-induced differentiationassociated protein 1), implique dans differentes formes de la maladie de Charcot-Marie-Tooth (CMT4A, cmT2K et AR-CMT2), code pour une proteine localisee sur la membrane externe mitochondriale. Les presentations phenotypiques des patients porteurs de mutations GDAP1 sont heterogenes ce qui rend difficile la determination de relations genotype-phenotype. Les bases de donnees specifiques de gene (LSDB), etablies dans le cadre du Projet Variome Humain, fournissent des outils puissants pour l’etude des maladies rares. Materiel et methodes C’est dans ce contexte que la Leiden Open-source Variation database (LOVD) a ete creee, dont le but est de developper un systeme simple de base de donnees pour la collecte des variations de sequence de genes en suivant les recommandations de la Human Genome Variation Society (HGVS). Nous avons utilise cet outil logiciel pour developper une base de donnees specifique dediee a GDAP1 ( http://mitodyn.org/ ). Cet outil permet de collecter toutes les variations de sequence pathogenes ou non pathogenes, publiees ou non publiees, en y associant des informations cliniques et electrophysiologiques, utiles pour le diagnostic moleculaire, les etudes statistiques et la determination de correlations genotype-phenotype dans cette maladie. Resultats Cette base de donnees contient actuellement 57 variants uniques rapportes chez 179 cas de cmT. L’analyse des donnees a permis de confirmer que les formes recessives sont plus severes que les formes dominantes. De plus, parmi les formes recessives, les mutations tronquantes sont responsables d’une evolution plus rapide de la maladie avec une dependance au fauteuil roulant quasi constante avant l’âge de 40 ans. Nous avons egalement montre l’importance des domaines Glutathion-S-transferases (GST), affectes dans la majorite des mutations, et associes a des phenotypes plus severes. Les donnees electrophysiologiques des patients inclus dans la base permettent de suggerer l’atteinte primitivement axonale de la maladie et l’absence de forme purement demyelinisante. Conclusion et perspectives A moyen terme, l’objectif est d’inclure dans notre base d’autres proteines impliquees dans la dynamique mitochondriale telles que OPA1, MFN2 et Drp1. Les maladies neurodegeneratives associees a ces genes sont prises en charge par les memes medecins et interessent les memes equipes de recherche.et il nous semble donc important de croiser les donnees genetiques et cliniques de ces patients afin d’ameliorer la strategie diagnostique de ces maladies.

V - 2 Propriétés électrophysiologies des souris NFHGFP : intérêt pour l’étude de la régénération axonale

Introduction Pour l’etude du metabolisme des neurofilaments, une lignee de souris transgeniques (NFHGFP), exprimant une proteine de fusion entre la sous-unite NFH et une proteine fluorescente (GFP) a ete creee (Letournel et al. , 2006). Objectifs L’etude veut demontrer que l’expression de la proteine modifiee n’altere pas les vitesses de conduction nerveuse (VCN) et que la queue peut etre utilisee pour une telle etude. Methodes Nous avons enregistre les VCN motrice des nerfs sciatiques des souris NFHGFP (n = 15) et de souris controles (n = 13), âgees de 3 a 4 mois. Dans le meme temps ont ete mesurees les vitesses de conduction motrice sur la queue en adaptant une technique decrite chez le rat. Un bloc de conduction a ensuite ete cree par ecrasement (« crush ») de la queue et du nerf sciatique, avec suivi de son evolution clinique et electrophysiologique. Resultats Aucune difference significative des VCN n’a ete constatee entre les souris NFHGFP et controles au niveau du nerf sciatique (40 m/s SEM ± 0,78 ; 42,7 m/s SEM ± 1,07) ou de la queue (30,6 m/s SEM ± 0,64 ; 32 m/s SEM ± 0,46). De plus le rapport entre vitesse de conduction sur la queue et sur le nerf sciatique est reste stable (0,76). Finalement l’aspect du bloc de conduction etait comparable entre les deux groupes. Discussion Cette etude montre que les VCN des souris NFHGFP et controles, et leur evolution lors de la regeneration nerveuse, sont similaires. Ces resultats montrent l’absence d’anomalie fonctionnelle des nerfs en accord avec l’absence d’anomalie morphologique deja rapportee. Les resultats obtenus par la methode de mesure des vitesses de conduction sur la queue des souris apparaissent plus reproductibles que ceux obtenus sur le nerf sciatique. Conclusion Les souris NFHGFP peuvent etre utilisees pour l’etude des Neurofilaments lors de la regeneration axonale. L’analyse des VCN sur la queue donne des resultats plus fiables que sur le nerf sciatique.