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We sought to improve the risk prediction of 3-month left ventricular remodeling (LVR) occurrence after myocardial infarction (MI), using a machine learning approach.Patients were included from a prospective cohort study analyzing the incidence of LVR in ST-elevation MI in 443 patients that were monitored at Angers University Hospital, France. Clinical, biological and cardiac magnetic resonance (CMR) imaging data from the first week post MI were collected, and LVR was assessed with CMR at 3 month. Data were processed with a machine learning pipeline using multiple feature selection algorithms to identify the most informative variables.We retrieved 133 clinical, biological and CMR imaging variables, from 379 patients with ST-elevation MI. A baseline logistic regression model using previously known variables achieved an AUC of 0.71 on the test set, with 67% sensitivity and 64% specificity. In comparison, our best predictive model was a neural network using seven variables (in order of importance): creatine kinase, mean corpuscular volume, baseline left atrial surface, history of diabetes, history of hypertension, red blood cell distribution width, and creatinine. This model achieved an AUC of 0.78 on the test set, reaching a sensitivity of 92% and a specificity of 55%, outperforming the baseline model.These preliminary results show the value of using an unbiased data-driven machine learning approach. We reached a higher level of sensitivity compared to traditional methods for the prediction of a 3-month post-MI LVR.

Purpose The lacrimal exocrinopathy of primary Sjogren's syndrome (pSS) is one of the main causes of severe dry eye syndrome and a burden for patients. Early recognition and treatment could prevent irreversible damage to lacrimal glands. The aim of this study was to find biomarkers in tears, using metabolomics and data mining approaches, in patients with newly-diagnosed pSS compared to other causes of dry eye syndrome. Methods A prospective cohort of 40 pSS and 40 non-pSS Sicca patients with dryness was explored through a standardized targeted metabolomic approach using liquid chromatography coupled with mass spectrometry. A metabolomic signature predictive of the pSS status was sought out using linear (logistic regression with elastic-net regularization) and non-linear (random forests) machine learning architectures, after splitting the studied population into training, validation and test sets. Results Among the 104 metabolites accurately measured in tears, we identified a discriminant signature composed of nine metabolites (two amino acids: serine, aspartate; one biogenic amine: dopamine; six lipids: Lysophosphatidylcholine C16:1, C18:1, C18:2, sphingomyelin C16:0 and C22:3, and the phoshatidylcholine diacyl PCaa C42:4), with robust performances (ROC-AUC = 0.83) for predicting the pSS status. Adjustment for age, sex and anti-SSA antibodies did not disrupt the link between the metabolomic signature and the pSS status. The non-lipidic components also remained specific for pSS regardless of the dryness severity. Conclusion Our results reveal a metabolomic signature for tears that distinguishes pSS from other dry eye syndromes and further highlight nine key metabolites of potential interest for early diagnosis and therapeutics of pSS.

Diseases caused by defects in mitochondrial DNA (mtDNA) maintenance machinery, leading to mtDNA deletions, form a specific group of disorders. However, mtDNA deletions also appear during aging, interfering with those resulting from mitochondrial disorders. Here, using next-generation sequencing (NGS) data processed by eKLIPse and data mining, we established criteria distinguishing age-related mtDNA rearrangements from those due to mtDNA maintenance defects. MtDNA deletion profiles from muscle and urine patient samples carrying pathogenic variants in nuclear genes involved in mtDNA maintenance (n = 40) were compared with age-matched controls (n = 90). Seventeen additional patient samples were used to validate the data mining model. Overall, deletion number, heteroplasmy level, deletion locations, and the presence of repeats at deletion breakpoints were significantly different between patients and controls, especially in muscle samples. The deletion number was significantly relevant in adults, while breakpoint repeat lengths surrounding deletions were discriminant in young subjects. Altogether, eKLIPse analysis is a powerful tool for measuring the accumulation of mtDNA deletions between patients of different ages, as well as in prioritizing novel variants in genes involved in mtDNA stability.

Mohassel et al. provide unprecedented dichotomy of consequences on sphingolipid biosynthesis between pathogenic variants in the SPTLC1 gene, responsible for either amyotrophic lateral sclerosis (ALS) or hereditary sensory and autonomic neuropathy type 1 (HSAN1). Normalization of sphingolipid levels by siRNA selectively targeting the ALS mutant allele mRNA sheds light on new therapeutic approaches.

Due to its pivotal role in NADH oxidation and ATP synthesis, mitochondrial complex I (CI) emerged as a crucial regulator of cellular metabolism. A functional CI relies on the sequential assembly of nuclear- and mtDNA-encoded subunits; however, whether CI assembly status is involved in the metabolic adaptations in CI deficiency still remains largely unknown. Here, we investigated the relationship between CI functions, its structure and the cellular metabolism in 29 patient fibroblasts representative of most CI mitochondrial diseases. Our results show that, contrary to the generally accepted view, a complex I deficiency does not necessarily lead to a glycolytic switch, i.e. the so-called Warburg effect, but that this particular metabolic adaptation is a feature of CI assembly defect. By contrast, a CI functional defect without disassembly induces a higher catabolism to sustain the oxidative metabolism. Mechanistically, we demonstrate that reactive oxygen species overproduction by CI assembly intermediates and subsequent AMPK-dependent Pyruvate Dehydrogenase inactivation are key players of this metabolic reprogramming. Thus, this study provides a two-way-model of metabolic responses to CI deficiencies that are central not only in defining therapeutic strategies for mitochondrial diseases, but also in all pathophysiological conditions involving a CI deficiency.

Introduction Mitochondrial diseases are a group of disorders caused mainly by the impairment of the mitochondrial oxidative phosphorylation process, due to mutations either in the mitochondrial or nuclear genome. Among them, the mitochondrial neuro-gastrointestinal encephalo-myopathy (MNGIE) syndrome affects adolescents or young adults, and is mostly caused by TYMP mutations encoding a cytosolic thymidine phosphorylase (TP). Patients and methods The present study reports the molecular investigation by next-generation re-sequencing of 281 nuclear genes, encoding mitochondrial proteins, of consanguineous family including two individuals with MNGIE syndrome associated to optic atrophy. Bioinformatic analysis was also performed in addition to mtDNA deletion screening and mtDNA copy number quantification in blood of the two patients which were carried out by solf clipping program and qPCR respectively. Results Next-generation re-sequencing revealed a novel homozygous c.2391G > T POLG mutation (p.M797I) co-occurring with the hypomorphic c.1311A > G OPA1 variant (p.I437M). Analysis of the mitochondrial genome in the two patients disclosed mtDNA depletion in blood, but no deletion. Bio-informatics investigations supported the pathogenicity of the novel POLG mutation that is located in the C-terminal subdomain and might change POLG 3D structure, stability and function. Conclusion The novel homozygous p.M797I POLG mutation is responsible for MNGIE combined to optic atrophy and mtDNA depletion in the two patients.

Resume Une hypothermie generalisee est une pathologie mettant en jeu le pronostic vital se definissant par une temperature corporelle inferieure a 35 °C. Son diagnostic post-mortem est delicat en raison de l’absence de signe pathognomonique. De nos jours, seule la presence simultanee de plusieurs arguments evocateurs, en l’absence d’element en faveur d’une autre etiologie, permet de porter son diagnostic. L’objectif de ce travail est de presenter un etat des lieux actualise des differents arguments diagnostiques macroscopiques, microscopiques et biologiques de la mort par hypothermie.

Resume Les maladies mitochondriales sont des pathologies frequentes du metabolisme caracterisees par une forte heterogeneite clinique et genetique. La double origine genetique des mitochondries, ADN nucleaire et ADN mitochondrial (ADNmt), et l’heteroplasmie mitochondriale, definie par la coexistence de mitochondries mutees et normales au sein des cellules, sont a l’origine de la complexite du diagnostic de ces maladies. La transmission de l’ADNmt est exclusivement maternelle, segregant de maniere aleatoire au cours des divisions cellulaires. Ces maladies mitochondriales peuvent survenir a n’importe quel âge de la vie et les presentations cliniques sont extremement variables touchant les tissus fortement consommateurs d’energie comme muscle ou cerveau mais tous les organes sont susceptibles d’etre atteints. Plusieurs centaines de mutations de l’ADNmt ont pu etre ainsi identifiees responsables de pathologies mitochondriales. Differents types de mutations de l’ADNmt, mutations ponctuelles, deletions ou depletion de l’ADNmt ont pu etre repertories. Le developpement des nouvelles technologies de sequeneage haut debit a considerablement ameliore le diagnostic permettant une analyse systematique de l’ADNmt avec une plus grande sensibilite dans la detection des variants. Cette analyse a revele en meme temps une plus grande complexite de l’analyse et interpretation des variants de l’ADNmt avec la necessite de developper de nouveaux outils bioinformatiques dedies a la genetique mitochondriale.

Introduction Entre 15 et 20 % de la thyroxine circulante (T4L) est transportee par la transthyretine codee par le gene TTR. Certains variants interferent avec les immunodosages des HT. Cette interference analytique mime le tableau biologique (TSH normale, T4L augmentee) retrouve dans les resistances aux hormones thyroidiennes (RHT). Patients et methodes Depuis 2016, 302 cas index ont ete adresses au centre de reference des maladies rares de la thyroide et des recepteurs hormonaux du CHU d’Angers pour suspicion de RHT. Les donnees clinico-biologiques ont ete collectees et nous avons realise le sequencage d’un panel de genes impliques dans les syndromes de sensibilite diminuee aux HT ainsi que celui du gene TTR (NM_000371,3). Resultats Sur ces 302 patients nous avons retrouve chez 29 (9,6 %) patients un variant du gene TTR. La majorite des patients (24 patients, 7,9 %) avait un variant non amyloidogene connu pour interferer avec les immunodosages (variant c.416C > T, p.[Thr139Met]). Chez les 5 autres patients : – 2 patients avaient le variant c.206C > T, p.Thr69Ile, variant amyloidogene connu sans description concernant l’impact sur le dosage des HT, – 3 patients avaient le variant c.424G > A, p.(Val142Ile), present a une frequence elevee (1,6 %) en population d’origine africaine et afro-americaine, responsable d’un sur-risque de cardiopathie et pouvant potentiellement alterer les dosages d’HT. Discussion Un variant du gene TTR doit faire partie des causes a eliminer devant une suspicion de RHT afin d’eviter les errances diagnostique et les traitements inadaptes. Il peut aussi permettre la decouverte d’une amylose hereditaire chez les patients.

Resume Une exploration biochimique post-mortem peut etre utile lorsque l’origine du deces n’a pas pu etre determinee par l’autopsie. Dans ce cadre, nous avons realise une revue de la litterature portant sur l’exploration post-mortem de l’acidocetose diabetique (ACD). But de l’etude Citer et developper les parametres utiles au diagnostic d’ACD en explorant leur evolution post-mortem. L’analyse simultanee du glucose, du D-β-hydroxybutyrate et de l’HbA 1c semble la plus contributive au diagnostic post-mortem d’ACD. L’exploration du glucose doit etre realisee au sein de l’humeur vitree avec un seuil de detection de l’hyperglycemie ante mortem fixe a 10 mmol/L. En effet, le glucose circulant peripherique n’est pas adapte, en lien avec une glycolyse anaerobie persistante entrainant une chute rapide de sa concentration. L’analyse du D-β-hydroxybutyrate, corps cetonique le plus specifique de l’ACD car ayant la plus grande stabilite post-mortem, peut etre realisee indifferemment sur le sang ou l’humeur vitree. Son seuil de detection de l’hypercetonemie ante mortem varie entre 2400 et 6000 μmol/L. Une HbA 1c superieure a 6,5 % (47,5 mmol/mol) permet de porter le diagnostic post-mortem de diabete. Une des limites de l’exploration de l’HbA 1c est sa stabilite estimee entre 36 et 72 heures. Le diabete peut egalement etre depiste par le dosage de la fructosamine circulante dont la stabilite est estimee a 72 heures. Conclusion Pour porter le diagnostic d’acidocetose diabetique, il est recommande d’analyser conjointement le glucose dans l’humeur vitree, le D-β-hydroxybutyrate et l’HbA 1c dans le sang peripherique. Cette revue de la litterature se base sur une recherche des articles references dans la base de donnees PubMed ® entre 1964 et aout 2016.

Resume Les maladies mitochondriales s’accompagnent frequemment d’une atteinte visuelle qui peut en constituer la manifestation inaugurale. Il peut s’agir d’une atteinte du nerf optique (neuropathie optique) ou encore d’une atteinte retinienne (retinopathie pigmentaire), des voies visuelles retro-chiasmatiques ou de l’oculomotricite. Les neuropathies optiques hereditaires les plus frequentes (neuropathie optique hereditaire de Leber et atrophie optique dominante), liees a un dysfonctionnement mitochondrial, peuvent etre isolees, ou peuvent s’associer a d’autres pathologies neurologiques. A l’inverse, plusieurs pathologies hereditaires et neurologiques syndromiques peuvent inclure dans leur tableau clinique une atteinte du nerf optique. Celle-ci s’explique par une vulnerabilite du nerf optique, qui dans sa partie intraoculaire est demyelinise, utilisant alors une grande quantite d’energie produite par les mitochondries pour la transmission de l’information visuelle. Plusieurs traitements pharmacologiques ou encore par therapie genique sont a l’etude, meme si pour l’instant, ces neuropathies mitochondriales restent incurables.

Introduction Le diagnostic de syndrome de Sjogren primaire (pSS) repose sur des criteres de classification comprenant des tests fonctionnels temoignant d’une atteinte lacrymale evoluee. Or, limiter la destruction des glandes lacrymales est un enjeu majeur pour reduire la morbidite du pSS. Ainsi l’identification de metabolites lacrymaux precocement perturbes pourrait servir de biomarqueur diagnostique. Quelques etudes ont pu identifier de potentiels biomarqueurs du syndrome sec oculaire mais aucune n’etait specifique du pSS. Les objectifs de cette etude etaient de rechercher une signature metabolomique des larmes chez des patients presentant un pSS nouvellement diagnostique, en comparaison a des patients presentant un syndrome sec oculaire non lie au pSS, et d’evaluer les variations de cette signature metabolomique selon la severite du syndrome sec oculaire. Patients et methodes Nous avons compare les compositions en metabolites des larmes de 40 patients pSS a 40 patients controles presentant un syndrome sec, par une approche metabolomique ciblee (Kit Biocrates® AbsoluteIDQ p180) selon une technique couplant chromatographie en phase liquide et spectrometre de masse (HPLC-MS/MS). Les larmes etaient recueillies sur bandelettes de test de Schirmer. Le plan d’analyse statistique comportait une etape de selection de variables par regression logistique avec regularisation de type elastic-net, puis une grid search comparant regression logistique et random forest. L’association de la signature metabolomique et du statut pSS etait ensuite analysee selon la severite du syndrome sec mesuree par le test de Schirmer, l’Ocular staining score (OSS) et la presence d’une dysfonction meibomienne. Les analyses statistiques ont ete menees avec les logiciels R software et SPSS. Le projet TEAR METABOGREN a fait l’objet d’un soutien financier de la part de l’association francaise du syndrome de Gougerot-Sjogren (AFGS) et de la SNFMI. Resultats Parmi les 188 metabolites testes, nous avons mis en evidence une signature metabolomique composee de 9 metabolites : Serine, Asparagine, Phosphatidylcholine diacyl (PCaa) C42 :4, lysophosphatidylcholines (LysoPC) C18 :1, C18 :2, C16 :1 et Sphingomyelines (SM) C16 :0 et C22 :3. Les capacites predictives de la signature metabolomique etaient interessantes (ROC-AUC 0,83) et restaient specifiques du pSS apres ajustement sur l’âge, le sexe, la presence d’anticorps anti-SSA et la severite du syndrome sec (test de Schirmer, presence d’une dysfonction meibomienne, break-up time test). Seule la composante lipidique de la signature variait selon le score OSS (p = 0,015). Conclusion La signature metabolomique identifiee au sein des larmes de patients pSS, en comparaison a des patients avec d’autres causes de syndromes secs pourrait servir de biomarqueur lacrymal precoce du pSS et identifier des pistes physiopathologiques pour l’elaboration de traitements specifiques.

Background: Intrauterine Growth Restriction (IUGR) affects 8% of newborns and increases morbidity and mortality for the offspring even during later stages of life. Omics studies have evidenced epigenetic, gene expression and metabolic alterations in IUGR, but pathogenic mechanisms as a whole are not being fully understood and integrated together. An in-depth strategy combining multi-omics integration could help to better understand these pathogenic processes. Methods: Methylomics and transcriptomics analyses were performed on 36 placenta samples in a case-control study. In addition to hierarchical clustering from the obtained quantitative data, machine learning algorithms were used to combine the analysis of more than 1200 genes found to be significantly modified. We used an automated text-mining approach, using the bulk textual gene annotations of the discriminant genes. The results were presented as clustered word clouds, allowing a rapid visualisation and summary of the pathophysiological processes involved. Support vector machine models were then used to explore the phenotypic subgroups (premature birth, birth weight and head circumference) associated with IUGR. Findings: Gene annotation clustering highlighted the alteration of cell signalling and proliferation, cytoskeleton and intra- and extracellular structures, oxidative stress, protein turnover, muscle development, energy and lipid metabolism with insulin resistance. Models obtained using a support vector machine showed a high capacity for predicting the sub-phenotypes associated with IUGR. Interpretation: Our results show that multi-omics integration, using the combination of gene annotation clustering, word cloud visualisation and support vector machine models, is highly relevant to provide an overview of the IUGR pathogenic mechanisms. Registration Number: The study was validated by the French CPP (Comite de Protection des Personnes) and registered to the French Ministry of Research under number DC-2009-907. Funding Statement: This study was funded by a grant from the University Hospital of Angers, France. Declaration of Interests: The authors state: "none declared." Ethics Approval Statement: All placentas and blood samples were collected from Angers University Hospital. This study was approved by the Ethics Committee of Angers. All patients gave their written consent for the use of their placenta. Clinical data related to the mother and the foetus, as well as neonatal data, were collected from the patients’ obstetric files. The cohort was registered at the French CNIL (Commission Nationale de l’Informatique et des Libertes no. pWP03752UL, ethics committee for the collection of clinical data from patient records).

International audience; Heme biosynthesis begins in the mitochondrion with the formation of delta-aminolevulinic acid (ALA). In acute intermittent porphyria, hereditary tyrosinemia type I and lead poisoning patients, ALA is accumulated in plasma and in organs, especially the liver. These diseases are also associated with neuromuscular dysfunction and increased incidence of hepatocellular carcinoma. Many studies suggest that this damage may originate from ALA-induced oxidative stress following its accumulation. Using theMnSOD as an oxidative stress marker, we showed here that ALA treatment of cultured cells induced ROS production, increasing with ALA concentration. The mitochondrial energetic function of ALA-treated HepG2 cells was further explored. Mitochondrial respiration and ATP content were reduced compared to control cells. For the 300μM treatment, ALA induced a mitochondrial mass decrease and a mitochondrial network imbalance although neither necrosis nor apoptosis were observed. The up regulation of PGC-1, Tfam and ND5 genes was also found; these genes encode mitochondrial proteins involved in mitochondrial biogenesis activation and OXPHOS function. We propose that ALA may constitute an internal bioenergetic signal, which initiates a coordinated upregulation of respiratory genes, which ultimately drives mitochondrial metabolic adaptation within cells. The addition of an antioxidant, Manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), resulted in improvement of maximal respiratory chain capacity with 300μM ALA. Our results suggest that mitochondria, an ALA-production site, are more sensitive to pro-oxidant effect of ALA, and may be directly involved in pathophysiology of patients with inherited or acquired porphyria.

International audience; This study of 200 Caucasian women shows that the distribution of the mtDNA macro-haplogroups in patients with diminished ovarian reserve (DOR) differed significantly from that of patients with normal ovarian reserve (NOR) (p=0.02). The JT macro-haplogroup was significantly under-represented in DOR patients compared with NOR patients (p=0.006) and compared with the estimated frequency of 18.8% in the general French population (p=0.0012). Our findings suggest that the risk of a prematurely depleted ovarian reserve would be three times lower for patients carrying the JT macro-haplogroup than for patients with any of the other mtDNA haplogroups (odds ratio: 0.3; 95% CI: 0.13-0.74). If these preliminary results are confirmed in larger independent studies, they should lead to the better management of infertility.

Resume Objectifs La diminution de la reserve ovarienne (DOR) est une cause courante d’infertilite. Nous avons cherche a definir les genes dont l’expression par les cellules de la corona radiata (CCR) pourrait etre specifiquement deregulee dans ce contexte. Patientes et methodes Dans le cadre de fecondations in vitro, nous avons analyse, a l’aide de microarrays, le profil d’expression differentiel des CCR chez 4 patientes presentant une DOR et chez 4 patientes presentant une reserve ovarienne normale. L’expression des genes d’interet a ensuite ete etudiee sur un panel independant de 40 patientes, par RT-PCR quantitative. Resultats Nous avons mis en evidence 48 transcrits differentiellement exprimes parmi lesquels, CXXC5 et FOXC1 sous-exprimes chez les patientes avec une DOR, et CTGF, FSTL3, PTGS2 et SOCS2 surexprimes dans ce groupe de patientes. Par ailleurs, deux sous-groupes ont ete individualises parmi les patientes presentant une DOR (DOR Gr1 et Gr2). Le groupe DOR Gr2 se caracterisait par une surexpression significative de CITED2, CTGF, GAS-1, IRS2, PTGS2, SOCS2, VCAN et une sous-expression significative de CXXC5, FOXC1, GBP2 et ZMIZ1. Onze de ces genes sont des cibles des oestrogenes et les patientes de ce groupe DOR2 presentaient un taux basal d’estradiol significativement plus eleve que les autres patientes du groupe DOR (p Discussion et conclusion Nous avons identifie 12 genes deregules dans les CCR de patientes presentant une DOR et potentiellement impliques dans ce phenotype. La distinction d’un sous-groupe particulier de DOR evoque la possibilite d’une deregulation des genes de reponse aux oestrogenes.

International audience; The m.3243A>G variant in the mitochondrial tRNA(Leu(UUR)) gene is a common mitochondrial DNA (mtDNA) mutation. Phenotypic manifestations depend mainly on the heteroplasmy, i.e. the ratio of mutant to normal mtDNA copies. A high percentage of mutant mtDNA is associated with a severe, life-threatening neurological syndrome known as MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). MELAS is described as a neurovascular disorder primarily affecting the brain and blood vessels, but the pathophysiology of the disease is poorly understood. We developed a series of cybrid cell lines at two different mutant loads: 70% and 100% in the nuclear background of a neuroblastoma cell line (SH-SY5Y). We investigated the impact of the mutation on the metabolism and mitochondrial respiratory chain activity of the cybrids. The m.3243A>G mitochondrial mutation induced a metabolic switch towards glycolysis in the neuronal cells and produced severe defects in respiratory chain assembly and activity. We used two strategies to compensate for the biochemical defects in the mutant cells: one consisted of lowering the glucose content in the culture medium, and the other involved the addition of l-arginine. The reduction of glucose significantly shifted the 100% mutant cells towards the wild-type, reaching a 90% mutant level and restoring respiratory chain complex assembly. The addition of l-arginine, a nitric oxide (NO) donor, improved complex I activity in the mutant cells in which the defective NO metabolism had led to a relative shortage of NO. Thus, metabolically induced heteroplasmy shifting and l-arginine therapy may constitute promising therapeutic strategies against MELAS.

International audience; IntroductionLes atrophies optiques héréditaires forment un groupe hétérogène de maladies responsables d’une dégénérescence des cellules ganglionnaires de la rétine avec une expression phénotypique variable, allant de formes asymptomatiques jusqu’à des formes sévères avec cécité. État des connaissances Les deux phénotypes les plus fréquents sont la maladie de Kjer, de transmission autosomique dominante provoquée par des mutations du gène OPA1 et la neuropathie optique héréditaire de Leber, causée par des mutations de l’ADN mitochondrial, de transmission maternelle. Perspectives et conclusion Ces deux neuropathies optiques habituellement isolées sont parfois associées à des signes extraoculaires essentiellement neurologiques centraux qui justifient un avis neurologique systématique ainsi qu’une imagerie cérébrale.

International audience; Ethambutol (EMB), widely used in the treatment of tuberculosis, has been reported to cause Leber’s 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.

Background Low levels of serum testosterone, as typically found in women and children, cannot be measured reliably by immunoassays. Our aim was to develop a sensitive assay to quantitate low serum testosterone concentrations using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results were compared to those obtained with various immunoassay techniques. Methods Serum testosterone levels in 70 women and children were measured using LC-MS/MS and compared with two automated, non-isotopic immunoassays, and three manual, isotopic immunoassays. Serum extraction was required only for LC-MS/MS and one of the isotopic methods. Results Deming regression analysis was used for comparison: the correlation coefficients were between 0.772 and 0.870, and the slopes between 0.972 and 1.365. Using Bland and Altman analysis, all the 5 immunoassays showed a positive mean difference compared with LC-MS/MS: all overestimated the testosterone levels in women and children. Conclusion None of the immunoassays tested proved sufficiently reliable when low testosterone concentrations (≤ 3.47 nmol/L) were measured. In contrast to conventional isotopic and non-isotopic immunoassay techniques, LC-MS/MS allows the precise determination of low testosterone levels. It has adequate sensitivity and is not subject to interference from other steroids that were tested.

Mitochondria play a primary role in cellular energetic metabolism, homeostasis and death. In spermatozoa, in particular, mitochondria produce the ATP necessary for motility. Mitochondrial functions depend, at least partially, on mitochondrial DNA (mtDNA). The mitochondrial genome, the transmission of which is exclusively maternal contributes to cytoplasmic heredity. Qualitative and quantitative mtDNA abnormalities have been associated with male infertility. This review focuses on the role of mtDNA in human fertility.

The mitochondria are dynamic organelles that constantly fuse and divide. An equilibrium between fusion and fission controls the morphology of the mitochondria, which appear as dots or elongated tubules depending the prevailing force. Characterization of the components of the fission and fusion machineries has progressed considerably, and the emerging question now is what role mitochondrial dynamics play in mitochondrial and cellular functions. Its importance has been highlighted by the discovery that two human diseases are caused by mutations in the two mitochondrial pro-fusion genes, MFN2 and OPA1. This review will focus on data concerning the function of OPA1, mutations in which cause optic atrophy, with respect to the underlying pathophysiological processes.

The Death Associated Protein 3 (DAP3), a GTP-binding constituent of the small subunit of the mitochondrial ribosome, is implicated in the TNFalpha and IFNgamma apoptotic pathways of the cell and is involved in the maintenance of the mitochondrial network. We have investigated the mitochondrial role of DAP3 by analyzing its mRNA and protein expression in transformed and non-transformed cell lines presenting various levels of mtDNA. The 3 mtDNA-less (rho degrees ) cell lines showed a complete absence of DAP3, whereas the mRNA expression was conserved. In HepG2 cells treated with increasing doses of ddCTP, the depletion of mtDNA was accompanied by the reduced expression of DAP3. However, the expression of the corresponding mRNA was maintained, suggesting the existence of a post-transcriptional mechanism responsible for the depletion of the DAP3. Compared to the parental cells, the 3 rho degrees cell lines displayed partial resistance to staurosporin-induced cell death. The absence of pro-apoptotic DAP3 in these mtDNA-less cells could explain their reduced apoptotic capacity. Our results suggest that the mtDNA content plays a role in cell apoptosis by mediating the expression of DAP3.

Resume Introduction En 1961, Garcin et collaborateurs publiaient dans la Revue Neurologique le cas de patients d’une meme famille atteints d’une atrophie optique associee a une cataracte et a des signes neurologiques. Les auteurs en faisaient une nouvelle entite clinique distincte des cadres nosologiques decrits a l’epoque (syndromes de Behr, Marinesco-Sjogren ou maladie de Friedreich). Methode Cette famille a ete suivie pendant 40 ans et le sequencage de differents genes responsables d’atrophies optiques a ete realise. Resultats La mutation G277A du gene OPA3 est responsable de la pathologie observee dans cette famille. Discussion Une nouvelle entite clinique est ainsi identifiee : l’atrophie optique et cataracte autosomique dominante provoquee par une mutation heterozygote du gene OPA3 , un gene nucleaire codant pour une proteine mitochondriale.

It has been shown previously that human rho degrees cells, deprived of mitochondrial DNA and consequently of functional oxidative phosphorylation, maintain a mitochondrial membrane potential, which is necessary for their growth. The goal of our study was to determine the precise origin of this membrane potential in three rho degrees cell lines originating from the human HepG2, 143B, and HeLa S3 cell lines. Residual cyanide-sensitive oxygen consumption suggests the persistence of residual mitochondrial respiratory chain activity, about 8% of that of the corresponding parental cells. The fluorescence emitted by the three rho degrees cell lines in the presence of a mitochondrial specific fluorochrome was partially reduced by a protonophore, suggesting the existence of a proton gradient. The mitochondrial membrane potential is maintained both by a residual proton gradient (up to 45 to 50% of the potential) and by other ion movements such as the glycolytic ATP(4-) to mitochondrial ADP(3-) exchange. The ANT2 gene, encoding isoform 2 of the adenine nucleotide translocator, is overexpressed in rho degrees HepG2 and 143B cells strongly dependent on glycolytic ATP synthesis, as compared to the corresponding parental cells, which present a more oxidative metabolism. In rho degrees HeLa S3 cells, originating from the HeLa S3 cell line, which already displays a glycolytic energy status, ANT2 gene expression was not higher as in parental cells. Mitochondrial oxygen consumption and ANT2 gene overexpression vary in opposite ways and this suggests that these two parameters have complementary roles in the maintenance of the mitochondrial membrane potential in rho degrees cells.

Nuclear mitochondrial pseudogenes (Numts) have been found in the genome of many eukaryote species, including humans. Using a BLAST approach, we found 1105 DNA sequences homologous to mitochondrial DNA (mtDNA) in the August 2001 Goldenpath human genome database. We assembled these sequences manually into 286 pseudogenes on the basis of single insertion events and constructed a chromosomal map of these Numts. Some pseudogenes appeared highly modified, containing inversions, deletions, duplications, and displaced sequences. In the case of four randomly selected Numts, we used PCR tests on cells lacking mtDNA to ensure that our technique was free from genome-sequencing artifacts. Furthermore, phylogenetic investigation suggested that one Numt, apparently inserted into the nuclear genome 25-30 million years ago, had been duplicated at least 10 times in various chromosomes during the course of evolution. Thus, these pseudogenes should be very useful in the study of ancient mtDNA and nuclear genome evolution.

Human DAP3 (death-associated protein-3) has been identified as an essential positive mediator of programmed cell death. Structure–function studies have shown previously the N-terminal extremity of the protein to be required in apoptosis induction. Analysis of human DAP3 gene structure predicted 13 exons and subsequent targeting prediction by two software packages (MITOPROT and TargetP) gave a high probability for mitochondrial targeting. The predicted N-terminal targeting structure was also found in the mouse, Drosophila, and C. elegans orthologues with a strong sequence homology between mouse and human. Secondary structure analyses identified α-helical structures typical of mitochondrial target peptides. To confirm experimentally this targeting we constructed a fusion protein with N-terminal human DAP3 upstream of enhanced green fluorescent protein (EGFP). Confocal analysis of transfected human fibroblasts clearly demonstrated EGFP localization exclusive to mitochondria. The positioning of this key apoptotic factor at the heart of the mitochondrial pathway provides exciting insight into its role in programmed cell death.

Multiple deletions of mtDNA have not only been implicated in aging, but also in a wide variety of pathological conditions. The enzyme system used in long-PCR makes it possible to synthesize the entire mitochondrial genome (16.5 kb), exposing the multiple deletions in mtDNAs implicated in and, at least partially, responsible for these pathologies. But it is not the number or type of anomalous mtDNA that is crucial, rather it is their frequency relative to the number of intact copies of the mitochondrial genome. Our work exposes the necessity of quantitating the number of normal mitochondrial DNAs. The accuracy of the technique and the small sample size required permit one to detect multiple deletions, located in a specific organ, and simultaneously measure the fraction of intact molecules. This fraction can then be correlated with mitochondrial dysfunction to serve both as an indicator of tissue aging and a monitor of an impending myopathy.

Background Remote ischemic preconditionning (RIPC) has emerged as an attractive therapeutic procedure to protect the heart against ischemia/reperfusion (I/R) injury. Despite strong evidences of the critical role played by circulating humoral mediators for signal transduction, their identities still remain unknown. Using a targeted metabolomic approach we previously identified Glycine (GLY) and Kynurenine (KYN) as potential mediators for RIPC induced cardioprotection in both rats and humans. Aim of the study this study sought to determine whether a treatment with GLY and KYN alone or in combination could mimic the cardioprotective effect of the RIPC. Methods Male Wistar rats, 8-10 weeks exposed to myocardial I/R were allocated to one of the following six groups: Myocardial Infarction (MI), rats were subjected to myocardial I/R without any further intervention; RIPC+MI, four cycles of limb I/R were applied prior to the myocardial ischemia; Metabolites+ MI, with intraperitoneal (ip) injection of a combined GLY (0.5mg/g body weight) and KYN (0.3 mg/g body weight) 10 min before MI; Vehicle+MI, with ip injection of the vehicle 10 min before MI; GLY+MI, with ip injection of single dose of GLY 10 min before MI; and KYN+MI, with an ip injection of single dose of KYN 10 min prior to MI. Hearts were excised after 2h of reperfusion, the area at risk (AAR) and infarct size (AN) were measured after TTC staining. GLY and KYN plasma concentrations were determined by mass spectroscopy LC-MS/MS at 3 points of time: 15, 30 and 45 min after the ip injection. Results The metabolites blood assay showed a significant increase of both glycine and kynurenine in blood plasma 15 min after the intraperitoneal injection with a peak at 30 min. As compared to MI, both RIPC+MI and Metabolites+MI exhibited a smaller infarct size (AN/AAR=39.59±3.76% for Vehicle+MIvs. 27.15±4.47% for the Metabolites+MI group, and 42.49±2.42% for MIvs. 34.53±2.28% for the RIPC+MI, both p Conclusion We report that RIPC-induced cardioprotection may involve a cocktail of amino acids for signal transduction from the remote organ to the myocardium. The author hereby declares no conflict of interest

We analyzed the mitochondrial DNA of patients with polymyalgia rheumatica, a disease frequently associated with mitochondrial myopathy. In an attempt to study the deletions, we have developed a qualitative PCR method using a highly thermostable polymerase in order to amplify multiple mitochondrial DNA large fragments (up to 12 kb). PCR serves to observe both deleted and normal fractions of the mitochondrial DNA. We found multiple deletions of the mitochondrial DNA in all of the patient muscles. Although these muscles harbored many ragged red fibers, we found no point mutations of the tRNA(Leu)(UUR)) and the mutation at nucleotide position 8344 was not present.

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.

Introduction Sept porteurs sains, appartenant a des familles atteintes de neuropathie optique hereditaire de Leber (NOHL) ont ete etudies. Materiels et Methodes Nous avons etudies les aspects cliniques familiaux, fonctionnels (acuites visuelles apres correction optique, etude de la vision des couleurs) ainsi que les aspects ophtalmoscopiques de la region papillaire et en OCT (epaisseur de la couche des fibres optiques). Resultats Mutation primaire preponderante :11 778. Tous les yeux ont une acuite visuelle au moins egale a 20/20. Plus de la moitie ont au fond d’œil des telangiectasies papillaires. Le test de la vision des couleurs permet de retrouver quelques anomalies (1 axe tetartan, 2 inversions sans axe) chez 3 patients mais sans aucun axe rouge-vert. Enfin la mesure de l’epaisseur des fibres nerveuses par OCT au niveau de la papille retrouve des valeurs elevees. Discussion Nous retenons l’apparente frequence des telangiectasies et/ou pseudo-œdeme, parfois une augmentation de l’epaisseur des fibres nerveuses et des troubles dyschromatopsiques. Si l’epaississement de la couche des fibres nerveuses semble integre a la pathogenie de la maladie, il est difficile de preciser s’il correspond a un stade subclinique ou bien a un stade preclinique. Conclusion Il n’est pas impossible s’il s’agit d’un stade subclinique que ces patients soient des « patients resistants ». Ils n’auraient dans ce cas pas plus de risques (et peut etre meme moins de risque) de developper une baisse d’acuite visuelle. Savini, Barboni et al., ophthalmology, 2005, 112(1) : 127-31 Retinal nerve fiber layer evaluation by OCT in unaffected carriers with Leber's hereditary optic neuropathy mutations. Ventura, Quiros et al., Invest Ophthalmol Vis Sci 2007, 48(5), 2362-70 Male prevalence of acquired color vision defects in asymptomatic carriers of Leber hereditary optic neuropathy.

Introduction Les atrophies optiques d’origine genetique sont parfois associees a des signes neurologiques centraux comme cela est decrit pour la maladie de Leber. Observations Patient ne en 1963 (dossier 060370), sans antecedent personnel ni familial. Il presenta en 2004 une baisse d’acuite visuelle bilaterale d’installation progressive rapidement associee a une nevralgie faciale gauche. En juin 2005, apparurent des douleurs du membre inferieur gauche, associees a des troubles proprioceptifs, et a un syndrome pyramidal reflexe du membre inferieur gauche, sans deficit moteur. L’examen ophtalmologique trouva une baisse d’acuite visuelle bilaterale avec scotome centro-caecal, une dyschromatopsie, une atteinte majeure des potentiels evoques visuels et une atrophie optique moderee bilaterale. La ponction lombaire identifia une hyperproteinorachie moderee (0,61 g/l), avec un aspect oligoclonal. L’IRM montra des hypersignaux T2 de la substance blanche peri ventriculaires ainsi que du tronc cerebral et de la moelle cervico-thoracique. L’atteinte bilaterale et simultanee des nerfs optiques justifia l’etude moleculaire des atrophies optiques genetiquement determinees. Aucune des 12 mutations caracteristiques de la maladie de Leber ne fut identifiee. En revanche, le sequencage du gene OPA1 montra la mutation C 1937 T a l’etat heterozygote. Discussion Plusieurs cas ont ete rapportes dans la litterature de patients atteints de troubles neurologiques centraux dans le cadre d’une maladie de Leber conduisant au concept de « maladie de Leber plus ». Le phenotype de notre patient est tout a fait comparable a ce qui est rapporte chez ces patients « Leber plus ». Nous proposons de definir comme « OPA1 plus » le phenotype associant atrophie optique, atteinte neurologique centrale et mutation du gene OPA1. Conclusion La decouverte d’une atrophie optique genetiquement determinee doit conduire a rechercher d’autres atteintes neurologiques eventuellement associees.

Introduction Les atrophies optiques (AO) mitochondriales incluent les atrophies optiques autosomiques dominantes (AOAD), la neuropathie optique hereditaire de Leber (NOHL) et l’atrophie optique avec cataracte autosomique dominante (AOCAD). Objectifs L’objectif est de determiner chez des patients atteints d’AO ceux porteurs de mutation soit des genes OPA1 (AOAD) ou OPA3 (AOCAD) soit d’une mutation associee a la NOHL et d’effectuer une correlation phenotype genotype. Methodes Des mutations des genes OPA1, OPA3 et les 12 mutations de l’ADN mitochondrial associees a la maladie de Leber furent recherchees par sequencage a partir des prelevements d’ADN adresses a notre laboratoire dans un contexte d’atrophie optique. Le phenotype des patients en fonction de leur genotype fut etabli a partir des fiches de renseignements cliniques joints aux prelevements. Resultats L’ADN de 600 patients atteints d’une AO fut etudie. 60 p. 100 etaient des cas familiaux, 40 p. 100 des cas sporadiques. Le plus souvent, le mode de transmission n’etait pas defini avec certitude. Une mutation fut identifiee sur l’ADN de 290 patients (49 p. 100). Une mutation de l’ADN mitochondrial fut identifiee sur l’ADN de 90 patients (15 p. 100), 180 patients (31 p. 100) avaient une mutation d’OPA1 et 14 patients (2 p. 100) une mutation d’OPA3. Certains patients atteints d’AOAD souffraient egalement de surdite ou de neuropathie axonale. Discussion Cette etude souligne l’interet d’effectuer des etudes moleculaires dans un contexte d’atrophie optique sans autre etiologie connue meme s’il n’y a pas d’antecedent familial identifie. De plus, ces patients doivent etre explores systematiquement sur le plan neurologique et audiologique. Conclusion L’atrophie optique d’origine genetique est parfois une maladie responsable d’un phenotype allant au-dela du nerf optique qu’il faut savoir rechercher.

Introduction Des inclusions intranucleaires diffuses d’Huntingtine polyglutaminee, debordant le striatum, sont la signature neuropathologique de l’expansion instable d’un triplet CAG sur le gene IT15 (chromosome 4). Objectifs Par l’analyse de differentes regions cerebrales nous recherchons l’existence d’une correlation entre le nombre d’inclusions et la quantite d’ADN mitochondrial. Methodes Pour trois cerveaux de patients atteints de maladie de Huntington et un cerveau controle nous avons selectionne et etudie (colorations standard et immunohistochimie : GFAP, polyglutamine) 11 regions representatives, incluant le striatum. La quantification a porte sur le nombre de neurones, d’inclusions intranucleaires, d’astrocytes et de l’ADN mitochondrial. Les resultats pour chaque item ont ete compares par un test non parametrique (logiciel JMP 6). Resultats Pour cette etude nous avons observe : que le nombre de CAG, compare aux resultats observes du vivant des patients, sur l’ADN lymphocytaire, ne variait pas dans le temps ni dans les differentes regions ; qu’il n’existait pas de correlation entre le nombre de CAG et le nombre d’inclusions qui variait selon les regions ; que la masse d’ADN mitochondrial bien que globalement diminuee, selon les regions, un nombre d’inclusions eleve allait de pair avec une moindre diminution de l’ADN mitochondrial. Discussion L’huntingtine mutee s’accompagne d’un deficit de la conversion energetique mitochondriale. On sait que la presence d’inclusions n’est pas correlee avec l’apoptose et est insuffisante pour la declencher. Nous demontrons egalement qu’un nombre eleve d’inclusions s’accompagne d’une meilleure conservation de la masse d’ADN mitochondriale. Contrairement a d’autres etudes, nous n’avons pas trouve de difference du nombre de CAG en fonction des regions. Conclusion Nos resultats confirment une stabilite du nombre de CAG, renforcent l’idee d’une implication mitochondriale et d’un role protecteur sur la mort neuronale des inclusions dans la maladie de Huntington.