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Hereditary optic neuropathies are caused by the degeneration of retinal ganglion cells whose axons form the optic nerves, with a consistent genetic heterogeneity. As part of our diagnostic activity, we retrospectively evaluated the combination of Leber hereditary optic neuropathy mutations testing with the exon sequencing of 87 nuclear genes on 2186 patients referred for suspected hereditary optic neuropathies. The positive diagnosis rate in individuals referred for Leber hereditary optic neuropathy testing was 18% (199/1126 index cases), with 92% (184/199) carrying one of the three main pathogenic variants of mitochondrial DNA (m.11778G>A, 66.5%; m.3460G>A, 15% and m.14484T>C, 11%). The positive diagnosis rate in individuals referred for autosomal dominant or recessive optic neuropathies was 27% (451/1680 index cases), with 10 genes accounting together for 96% of this cohort. This represents an overall positive diagnostic rate of 30%. The identified top 10 nuclear genes included OPA1, WFS1, ACO2, SPG7, MFN2, AFG3L2, RTN4IP1, TMEM126A, NR2F1 and FDXR. Eleven additional genes, each accounting for less than 1% of cases, were identified in 17 individuals. Our results show that 10 major genes account for more than 96% of the cases diagnosed with our nuclear gene panel.

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.

Mitochondrial cytopathies, among which the Leigh syndrome (LS), are caused by variants either in the mitochondrial or the nuclear genome, affecting the oxidative phosphorylation process. The aim of the present study consisted in defining the molecular diagnosis of a group of Tunisian patients with LS. Six children, belonging to five Tunisian families, with clinical and imaging presentations suggestive of LS were recruited. Whole mitochondrial DNA and targeted next-generation sequencing of a panel of 281 nuclear genes involved in mitochondrial physiology were performed. Bioinformatic analyses were achieved in order to identify deleterious variations. A single m.10197G>A (p.Ala47Thr) variant was found in the mitochondrial MT-ND3 gene in one patient, while the others were related to autosomal homozygous variants: two c.1412delA (p.Gln471ArgfsTer42) and c.1264A>G (p.Thr422Ala) in SLC19A3, one c.454C>G (p.Pro152Ala) in SLC25A19 and one c.122G>A (p.Gly41Asp) in ETHE1. Our findings demonstrate the usefulness of genomic investigations to improve LS diagnosis in consanguineous populations and further allow for treating the patients harboring variants in SLC19A3 and SLC25A19 that contribute to thiamine transport, by thiamine and biotin supplementation. Considering the Tunisian genetic background, the newly identified variants could be screened in patients with similar clinical presentation in related populations.

BackgroundMitochondrial disorders (MD) are metabolic diseases related to genetic mutations in mitochondrial DNA and nuclear DNA that cause dysfunction of the mitochondrial respiratory chain. Cognitive impairment and psychiatric symptoms are frequently associated with MD in the adult population. The aim of this study is to describe the neuropsychological profile in children and adolescents with MD.MethodsWe prospectively recruited a sample of 12 children and adolescents between February 2019 and February 2020 in the Reference Center for Mitochondrial Disorders of Angers (France). Participants and their parents completed an anamnestic form describing socio-demographic data and completed the WISC-V (Wechsler Intelligence Scale for Children, 5th edition) and the Parent and Teacher forms of the BRIEF (Behavior Rating Inventory of Executive Function).ResultsIn our sample, the mean IQ (Intellectual Quotient) score was 87.3 ± 25.3. The score ranged from 52 to 120. Concerning executive functions, a significant global clinical complaint was found for parents (six times more than normal) and to a lesser extent, for teachers (among 3 to 4 times more). Levels of intelligence and executive functioning were globally linked in our cohort but dissociation remains a possibility.ConclusionThe results of this study show that MD can be associated to neuropsychological disorders in children and adolescents, especially regarding the intelligence quotient and the executive function. Our study also highlights the need for regular neuropsychological assessments in individuals with MD and developing brains, such as children and adolescents.

Inherited optic neuropathies are the most common mitochondrial diseases, leading to neurodegeneration involving the irreversible loss of retinal ganglion cells, optic nerve degeneration and central visual loss. Importantly, properly regulated mitochondrial dynamics are critical for maintaining cellular homeostasis, and are further regulated by MIEF1 (mitochondrial elongation factor 1) which encodes for MID51 (mitochondrial dynamics protein 51), an outer mitochondrial membrane protein that acts as an adaptor protein to regulate mitochondrial fission. However, dominant mutations in MIEF1 have not been previously linked to any human disease. Using targeted sequencing of genes involved in mitochondrial dynamics, we report the first heterozygous variants in MIEF1 linked to disease, which cause an unusual form of late-onset progressive optic neuropathy characterized by the initial loss of peripheral visual fields. Pathogenic MIEF1 variants linked to optic neuropathy do not disrupt MID51’s localization to the outer mitochondrial membrane or its oligomerization, but rather, significantly disrupt mitochondrial network dynamics compared to wild-type MID51 in high spatial and temporal resolution confocal microscopy live imaging studies. Together, our study identifies dominant MIEF1 mutations as a cause for optic neuropathy and further highlights the important role of properly regulated mitochondrial dynamics in neurodegeneration.

Although RTN4IP1 has been firmly associated to recessive optic atrophies, we identified by systematic examination of the 55° fundus autofluorescence frames and the peripheral spectral-domain optical coherence tomographic images, a progressive rod–cone dystrophy in all individuals with biallelic RTN4IP1 mutations., Purpose: RTN4IP1 biallelic mutations cause a recessive optic atrophy, sometimes associated to more severe neurological syndromes, but so far, no retinal phenotype has been reported in RTN4IP1 patients, justifying their reappraisal. Methods: Seven patients from four families carrying biallelic RTN4IP1 variants were retrospectively reviewed, with emphasis on their age of onset, visual acuity, multimodal imaging including color and autofluorescence frames, spectral-domain optical coherence tomography with RNFL and macular analyses. Results: Seven patients from four RTN4IP1 families developed in their first decade of life a bilateral recessive optic atrophy with severe central visual loss, and primary nystagmus developed in 5 of 7 patients. Six patients were legally blind. In a second stage, the seven individuals developed a rod–cone dystrophy, sparing the macular zone and the far periphery. This retinal damage was identified by 55° field fundus autofluorescence frames and also by spectral-domain optical coherence tomography scans of the temporal part of the macular zone in five of the seven patients. Full-field electroretinography measurements disclosed reduced b-wave amplitude of the rod responses in all patients but two. Family 4 with the p.R103H and c.601A > T (p.K201*) truncating mutation had further combined neurological signs with cerebellar ataxia, seizures, and intellectual disability. Conclusion: RTN4IP1 recessive optic atrophy is systematically associated to a rod–cone dystrophy, which suggests that both the retinal ganglion cells and the rods are affected as a result of a deficit in the mitochondrial respiratory chain. Thus, systematic widefield autofluorescence frames and temporal macular scans are recommended for the evaluation of patients with optic neuropathies.

Background: FOXL2 is the gene involved in blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). There have been few single case reports of growth hormone deficiency (GHD) with this syndrome, and Foxl2 is known to be involved in pituitary development in mice. Our aim was to analyze the prevalence of FOXL2 gene alteration in a series of patients with congenital hypopituitarism and eyelid anomalies. Methods: FOXL2 was analyzed in 10 patients with hypopituitarism (ranging from isolated GHD to complete pituitary hormone deficiency) and eyelid anomalies (typical BPES in 4 patients and milder anomalies in 6 patients). In patients with an FOXL2 mutation, we ruled out other possible molecular explanations by analyzing a panel of 20 genes known to be associated with hypopituitarism, and a candidate gene approach was used for patients without an FOXL2mutation. Results: Three patients had an FOXL2mutation. All 3 had typical BPES. Their pituitary phenotype varied from GHD to complete pituitary hormone deficiency and their pituitary morphology ranged from normal to an interrupted pituitary stalk. No mutations were found in genes previously associated with hypopituitarism. Conclusion: Our study shows that some patients with BPES have hypopituitarism with no molecular explanation other than FOXL2 mutation. This points toward an involvement of FOXL2 in human pituitary development.

Pathogenic variants of the nuclear receptor subfamily 2 group F member 1 gene (NR2F1) are responsible for Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), an autosomal dominant disorder characterized by optic atrophy associated with developmental delay and intellectual disability, but with a clinical presentation which appears to be multifaceted. We created the first public locus-specific database (LSDB) dedicated to NR2F1. All variants and clinical cases reported in the literature, as well as new unpublished cases, were integrated into the database using standard nomenclature to describe both molecular and phenotypic anomalies. We subsequently pursued a comprehensive approach based on computed representation and analysis suggesting a refinement of the BBSOAS clinical description with respect to neurological features and the inclusion of additional signs of hypotonia and feeding difficulties. This database is fully accessible for both clinician and molecular biologists and should prove useful in further refining the clinical synopsis of NR2F1 as new data is recorded. This article is protected by copyright. All rights reserved.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells., Graphical Abstract Graphical Abstract, By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, Charif et al. report 61 new cases harbouring variants in ACO2, among whom 50 with dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy.

Pathogenic variants of the aconitase 2 gene (ACO2) are responsible for a broad clinical spectrum involving optic nerve degeneration, ranging from isolated optic neuropathy with recessive or dominant inheritance, to complex neurodegenerative syndromes with recessive transmission. We created the first public locus-specific database (LSDB) dedicated to ACO2 within the “Global Variome shared LOVD” using exclusively the Human Phenotype Ontology (HPO), a standard vocabulary for describing phenotypic abnormalities. All the variants and clinical cases listed in the literature were incorporated into the database, from which we produced a dataset. We followed a rational and comprehensive approach based on the HPO thesaurus, demonstrating that ACO2 patients should not be classified separately between isolated and syndromic cases. Our data highlight that certain syndromic patients do not have optic neuropathy and provide support for the classification of the recurrent pathogenic variants c.220C>G and c.336C>G as likely pathogenic. Overall, our data records demonstrate that the clinical spectrum of ACO2 should be considered as a continuum of symptoms and refines the classification of some common variants., Measurement(s) sequence_variant • Phenotypic variability Technology Type(s) DNA sequencing • Ophthalmologist Factor Type(s) sequence variant • phenotype Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.13574528

Pathogenic variants of the nuclear receptor subfamily 2 group F member 1 gene (NR2F1) are responsible for Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), an autosomal dominant disorder characterized by optic atrophy associated with developmental delay and intellectual disability, but with a clinical presentation which appears to be multifaceted. We created the first public locus-specific database (LSDB) dedicated to NR2F1. All variants and clinical cases reported in the literature, as well as new unpublished cases, were integrated into the database using standard nomenclature to describe both molecular and phenotypic anomalies. We subsequently pursued a comprehensive approach based on computed representation and analysis suggesting a refinement of the BBSOAS clinical description with respect to neurological features and the inclusion of musculoskeletal hypotonia and intestinal signs with feeding difficulties. This database is fully accessible for both clinician and molecular biologists and should prove useful in further refining the clinical synopsis of NR2F1 as new data is recorded.

Biallelic mutations in ACO2, encoding the mitochondrial aconitase 2, have been identified in individuals with neurodegenerative syndromes, including infantile cerebellar retinal degeneration and recessive optic neuropathies (locus OPA9). By screening European cohorts of individuals with genetically unsolved inherited optic neuropathies, we identified 61 cases harbouring variants in ACO2, among whom 50 carried dominant mutations, emphasizing for the first time the important contribution of ACO2 monoallelic pathogenic variants to dominant optic atrophy. Analysis of the ophthalmological and clinical data revealed that recessive cases are affected more severely than dominant cases, while not significantly earlier. In addition, 27% of the recessive cases and 11% of the dominant cases manifested with extraocular features in addition to optic atrophy. In silico analyses of ACO2 variants predicted their deleterious impacts on ACO2 biophysical properties. Skin derived fibroblasts from patients harbouring dominant and recessive ACO2 mutations revealed a reduction of ACO2 abundance and enzymatic activity, and the impairment of the mitochondrial respiration using citrate and pyruvate as substrates, while the addition of other Krebs cycle intermediates restored a normal respiration, suggesting a possible short-cut adaptation of the tricarboxylic citric acid cycle. Analysis of the mitochondrial genome abundance disclosed a significant reduction of the mitochondrial DNA amount in all ACO2 fibroblasts. Overall, our data position ACO2 as the third most frequently mutated gene in autosomal inherited optic neuropathies, after OPA1 and WFS1, and emphasize the crucial involvement of the first steps of the Krebs cycle in the maintenance and survival of retinal ganglion cells.

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.

Pathogenic NR2F1 variants cause a rare autosomal dominant neurodevelopmental disorder referred to as the Bosch–Boonstra–Schaaf Optic Atrophy Syndrome. Although visual loss is a prominent feature seen in affected individuals, the molecular and cellular mechanisms contributing to visual impairment are still poorly characterized. We conducted a deep phenotyping study on a cohort of 22 individuals carrying pathogenic NR2F1 variants to document the neurodevelopmental and ophthalmological manifestations, in particular the structural and functional changes within the retina and the optic nerve, which have not been detailed previously. The visual impairment became apparent in early childhood with small and/or tilted hypoplastic optic nerves observed in 10 cases. High-resolution optical coherence tomography imaging confirmed significant loss of retinal ganglion cells with thinning of the ganglion cell layer, consistent with electrophysiological evidence of retinal ganglion cells dysfunction. Interestingly, for those individuals with available longitudinal ophthalmological data, there was no significant deterioration in visual function during the period of follow-up. Diffusion tensor imaging tractography studies showed defective connections and disorganization of the extracortical visual pathways. To further investigate how pathogenic NR2F1 variants impact on retinal and optic nerve development, we took advantage of an Nr2f1 mutant mouse disease model. Abnormal retinogenesis in early stages of development was observed in Nr2f1 mutant mice with decreased retinal ganglion cell density and disruption of retinal ganglion cell axonal guidance from the neural retina into the optic stalk, accounting for the development of optic nerve hypoplasia. The mutant mice showed significantly reduced visual acuity based on electrophysiological parameters with marked conduction delay and decreased amplitude of the recordings in the superficial layers of the visual cortex. The clinical observations in our study cohort, supported by the mouse data, suggest an early neurodevelopmental origin for the retinal and optic nerve head defects caused by NR2F1 pathogenic variants, resulting in congenital vision loss that seems to be non-progressive. We propose NR2F1 as a major gene that orchestrates early retinal and optic nerve head development, playing a key role in the maturation of the visual system., Jurkute et al. expands the phenotypic spectrum of pathogenic NR2F1 variants, providing a detailed characterization of the retinal and optic nerve changes with high-resolution optical coherence tomography imaging. Data from a mutant mouse model confirms the key role played by NR2F1 in directing retinogenesis and the development of the optic nerve head., Graphical Abstract Graphical Abstract

Leber’s hereditary optic neuropathy (LHON) is the most common disorder due to mitochondrial DNA mutations and complex I deficiency. It is characterized by an acute vision loss, generally in young adults, with a higher penetrance in males. How complex I dysfunction induces the peculiar LHON clinical presentation remains an unanswered question. To gain an insight into this question, we carried out a non-targeted metabolomic investigation using the plasma of 18 LHON patients, during the chronic phase of the disease, comparing them to 18 healthy controls. A total of 500 metabolites were screened of which 156 were accurately detected. A supervised Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) highlighted a robust model for disease prediction with a Q2 (cum) of 55.5%, with a reliable performance during the permutation test (cross-validation analysis of variance, P-value = 5.02284e−05) and a good prediction of a test set (P = 0.05). This model highlighted 10 metabolites with variable importance in the projection (VIP) > 0.8. Univariate analyses revealed nine discriminating metabolites, six of which were the same as those found in the Orthogonal Projections to Latent Structures Discriminant Analysis model. In total, the 13 discriminating metabolites identified underlining dietary metabolites (nicotinamide, taurine, choline, 1-methylhistidine and hippurate), mitochondrial energetic substrates (acetoacetate, glutamate and fumarate) and purine metabolism (inosine). The decreased concentration of taurine and nicotinamide (vitamin B3) suggest interesting therapeutic targets, given their neuroprotective roles that have already been demonstrated for retinal ganglion cells. Our results show a reliable predictive metabolomic signature in the plasma of LHON patients and highlighted taurine and nicotinamide deficiencies.

Background: Mitochondrial disorders (MD) are a group of clinically heterogeneous genetic disorders resulting from dysfunction of the mitochondrial respiratory chain. Cognitive impairment is a common feature in adults with MD and psychiatric symptoms are associated with MD in up to 70% of the adult population. The aim of this study is to describe the psychiatric profile in children and adolescents with MD by focusing on the description of psychiatric symptoms.Methods: A cohort of 12 children and adolescents was prospectively recruited between February 2019 and February 2020 in the Reference Center for Mitochondrial Disorders of Angers (France). Participants and their parents completed an anamnestic form to provide socio-demographic data and completed the Global Assessment of Functioning scale, the Brief Psychiatric Rating Scale, the Child Depression Inventory, the Revised Children's Manifest Anxiety Scale, and the Conner's Rating Scale to evaluate the inattention/hyperactivity symptoms as well as the Quality of Life scale.Results: Four children (33.3%) were diagnosed with depressive symptoms. With regarding to anxiety, 6 children (50%) reported anxiety issues during the psychiatric interview and 3 children (25%) were suffering from anxiety according to the RCMAS scale. Compared to other children with chronic illnesses, the individuals in our cohort reported a lower overall quality of life score and lower scores in physical and social subscales.Conclusion: Our study shows that MD can lead to psychiatric disorders in children and adolescents, in particular anxiety and depression, as well as poor quality of life. This highlights the need for regular psychiatric assessments in individuals with developing brains, such as children and adolescents. We do not, however, have data regarding the neuropsychological profile of this population.

Mitochondrial diseases are a clinically heterogeneous group of multisystemic disorders that arise as a result of various mitochondrial dysfunctions. Autosomal recessive aARS deficiencies represent a rapidly growing group of severe rare inherited mitochondrial diseases, involving multiple organs, and currently without curative option. They might be related to defects of mitochondrial aminoacyl t-RNA synthetases (mtARS) that are ubiquitous enzymes involved in mitochondrial aminoacylation and the translation process. Here, using NGS analysis of 281 nuclear genes encoding mitochondrial proteins, we identified 4 variants in different mtARS in three patients from unrelated Tunisian families, with clinical features of mitochondrial disorders. Two homozygous variants were found in KARS (c.683C>T) and AARS2 (c.1150-4C>G), respectively in two patients, while two heterozygous variants in EARS2 (c.486-7C>G) and DARS2 (c.1456C>T) were concomitantly found in the third patient. Bio-informatics investigations predicted their pathogenicity and deleterious effects on pre-mRNA splicing and on protein stability. Thus, our results suggest that mtARS mutations are common in Tunisian patients with mitochondrial diseases.

International audience

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.

FOXL2 is the gene involved in blepharophimosis, ptosis, and epicanthus inversus syndrome (BPES). There have been few single case reports of growth hormone deficiency (GHD) with this syndrome, and Foxl2 is known to be involved in pituitary development in mice. Our aim was to analyze the prevalence of FOXL2 gene alteration in a series of patients with congenital hypopituitarism and eyelid anomalies.FOXL2 was analyzed in 10 patients with hypopituitarism (ranging from isolated GHD to complete pituitary hormone deficiency) and eyelid anomalies (typical BPES in 4 patients and milder anomalies in 6 patients). In patients with an FOXL2 mutation, we ruled out other possible molecular explanations by analyzing a panel of 20 genes known to be associated with hypopituitarism, and a candidate gene approach was used for patients without an FOXL2mutation.Three patients had an FOXL2mutation. All 3 had typical BPES. Their pituitary phenotype varied from GHD to complete pituitary hormone deficiency and their pituitary morphology ranged from normal to an interrupted pituitary stalk. No mutations were found in genes previously associated with hypopituitarism.Our study shows that some patients with BPES have hypopituitarism with no molecular explanation other than FOXL2 mutation. This points toward an involvement of FOXL2 in human pituitary development.

Dominant optic atrophy is a blinding disease due to the degeneration of the retinal ganglion cells, the axons of which form the optic nerves. In most cases, the disease is caused by mutations in OPA1, a gene encoding a mitochondrial large GTPase involved in cristae structure and mitochondrial network fusion. Using exome sequencing, we identified dominant mutations in DNM1L on chromosome 12p11.21 in three large families with isolated optic atrophy, including the two families that defined the OPA5 locus on chromosome 19q12.1-13.1, the existence of which is denied by the present study. Analyses of patient fibroblasts revealed physiological abundance and homo-polymerization of DNM1L, forming aggregates in the cytoplasm and on highly tubulated mitochondrial network, whereas neither structural difference of the peroxisome network, nor alteration of the respiratory machinery was noticed. Fluorescence microscopy of wild-type mouse retina disclosed a strong DNM1L expression in the ganglion cell layer and axons, and comparison between 3-month-old wild-type and Dnm1l+/- mice revealed increased mitochondrial length in retinal ganglion cell soma and axon, but no degeneration. Thus, our results disclose that in addition to OPA1, OPA3, MFN2, AFG3L2 and SPG7, dominant mutations in DNM1L jeopardize the integrity of the optic nerve, suggesting that alterations of the opposing forces governing mitochondrial fusion and fission, similarly affect retinal ganglion cell survival.

Item does not contain fulltext BACKGROUND: Non-syndromic hereditary optic neuropathy (HON) has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function. However, the disease causing gene remains unknown in many families. The objective of the present study was to identify the molecular cause of non-syndromic LHON-like disease in siblings born to non-consanguineous parents of French origin. METHODS: We used a combination of genetic analysis (gene mapping and whole-exome sequencing) in a multiplex family of non-syndromic HON and of functional analyses in patient-derived cultured skin fibroblasts and the yeast Yarrowia lipolytica. RESULTS: We identified compound heterozygote NDUFS2 disease-causing mutations (p.Tyr53Cys; p.Tyr308Cys). Studies using patient-derived cultured skin fibroblasts revealed mildly decreased NDUFS2 and complex I abundance but apparently normal respiratory chain activity. In the yeast Y. lipolytica ortholog NUCM, the mutations resulted in absence of complex I and moderate reduction in nicotinamide adenine dinucleotide-ubiquinone oxidoreductase activity, respectively. CONCLUSIONS: Biallelism for NDUFS2 mutations causing severe complex I deficiency has been previously reported to cause Leigh syndrome with optic neuropathy. Our results are consistent with the view that compound heterozygosity for severe and hypomorphic NDUFS2 mutations can cause non-syndromic HON. This observation suggests a direct correlation between the severity of NDUFS2 mutations and that of the disease and further support that there exist a genetic overlap between non-syndromic and syndromic HON due to defective mitochondrial function.

Accurate detection of mitochondrial DNA (mtDNA) alterations is essential for the diagnosis of mitochondrial diseases. The development of high-throughput sequencing technologies has enhanced the detection sensitivity of mtDNA pathogenic variants, but the detection of mtDNA rearrangements, especially multiple deletions, is still poorly processed. Here, we present eKLIPse, a sensitive and specific tool allowing the detection and quantification of large mtDNA rearrangements from single and paired-end sequencing data. The methodology was first validated using a set of simulated data to assess the detection sensitivity and specificity, and second with a series of sequencing data from mitochondrial disease patients carrying either single or multiple deletions, related to pathogenic variants in nuclear genes involved in mtDNA maintenance. eKLIPse provides the precise breakpoint positions and the cumulated percentage of mtDNA rearrangements at a given gene location with a detection sensitivity lower than 0.5% mutant. eKLIPse software is available either as a script to be integrated in a bioinformatics pipeline, or as user-friendly graphical interface to visualize the results through a Circos representation ( https://github.com/dooguypapua/eKLIPse ). Thus, eKLIPse represents a useful resource to study the causes and consequences of mtDNA rearrangements, for further genotype/phenotype correlations in mitochondrial disorders.

International audience; We compared the metabolomic profile of aqueous humor from patients with primary open-angle glaucoma (POAG; n = 26) with that of a group of age- and sex-matched non-POAG controls (n = 26), all participants undergoing cataract surgery. Supervised paired partial least-squares discriminant analysis showed good predictive performance for test sets with a median area under the receiver operating characteristic of 0.89 and a p-value of 0.0087. Twenty-three metabolites allowed discrimination between the two groups. Univariate analysis after the Benjamini–Hochberg correction showed significant differences for 13 of these metabolites. The POAG metabolomic signature indicated reduced concentrations of taurine and spermine and increased concentrations of creatinine, carnitine, three short-chain acylcarnitines, 7 amino acids (glutamine, glycine, alanine, leucine, isoleucine, hydroxyl-proline, and acetyl-ornithine), 7 phosphatidylcholines, one lysophosphatidylcholine, and one sphingomyelin. This suggests an alteration of metabolites involved in osmoprotection (taurine and creatinine), neuroprotection (spermine, taurine, and carnitine), amino acid metabolism (7 amino acids and three acylcarnitines), and the remodeling of cell membranes drained by the aqueous humor (hydroxyproline and phospholipids). Five of these metabolic alterations, already reported in POAG plasma, concern spermine, C3 and C4 acylcarnitines, PC aa 34:2, and PC aa 36:4, thus highlighting their importance in the pathogenesis of glaucoma.

International audience; BACKGROUND: Myoclonic epilepsy with ragged-red fibers (MERRF, OMIM, #545000) is a rare neurological condition mostly caused by the m.8344A>G mitochondrial DNA pathogenic variant, which can variably affect multiple tissues, including the retina and optic nerve. We report detection of visually asymptomatic neuroretinal loss in 3 patients with genetically confirmed MERRF, using spectral domain optical coherence tomography (SD-OCT).METHODS: All patients underwent a complete ophthalmic examination including assessments of visual acuity, color vision, pupillary reactions, extraocular movements, applanation tonometry, slit-lamp, and dilated fundus examinations. Standard automated perimetry or Goldmann kinetic perimetry was performed, as well as fundus photographs and SD-OCT of the optic nerve head and macula.RESULTS: Despite the absence of visual symptoms in all patients, and normal visual acuity and visual fields in 1 patient, the 3 genetically confirmed patients (point mutations m.8344A>G; age range: 18-62 years) with MERRF-related neurological manifestations, displayed thinning of the retinal nerve fiber layer and variable alterations of the macular ganglion cell complex.CONCLUSIONS: Visually asymptomatic patients with genetically confirmed MERRF can display features of structural neuroretinal loss, quantifiable with SD-OCT. Further investigations are needed to establish whether OCT can assess early neurodegeneration in MERRF.

International audience; Mutations in the Optic Atrophy 1 gene (OPA1) were first identified in 2000 as the main cause of Dominant Optic Atrophy, a disease specifically affecting the retinal ganglion cells and the optic nerve. Since then, an increasing number of symptoms involving the central, peripheral and autonomous nervous systems, with considerable variations of age of onset and severity, have been reported in OPA1 patients. This variety of phenotypes is attributed to differences in the effects of OPA1 mutations, to the mode of inheritance, which may be mono- or bi-allelic, and eventually to somatic mitochondrial DNA mutations. The diversity of the pathophysiological mechanisms involved in OPA1-related disorders is linked to the crucial role played by OPA1 in the maintenance of mitochondrial structure, genome and function. The neurological expression of these disorders highlights the importance of mitochondrial dynamics in neuronal processes such as dendritogenesis, axonal transport, and neuronal survival. Thus, OPA1-related disorders may serve as a paradigm in the wider context of neurodegenerative syndromes, particularly for the development of novel therapeutic strategies against these diseases.

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.

International audience; Purpose: To determine the plasma metabolomic signature of primary open-angle glaucoma (POAG).Methods: We compared the metabolomic profiles of plasma from individuals with POAG (n = 36) with age- and sex-matched controls with cataract (n = 27). A targeted metabolomics study was performed using the standardized p180 Biocrates Absolute IDQ p180 kit with a QTRAP 5500 mass spectrometer. Multivariate analyses were performed using principal component analysis (PCA) and the least absolute shrinkage and selection operator (LASSO) method.Results: Among the 151 metabolites accurately measured, combined univariate and multivariate analyses revealed 18 discriminant metabolites belonging to the carbohydrate, acyl-carnitine, phosphatidylcholine, amino acids, and polyamine families. The metabolomic signature of POAG points to three closely interdependent pathophysiologic conditions; that is, defective mitochondrial oxidation of energetic substrates, altered metabolism resembling that observed in senescence, and a deficiency in spermidine and spermine, both polyamines being involved in the protection of retinal ganglion cells.Conclusions: Our results highlight a systemic and age-related mitochondrial defect in the pathogenesis of POAG.

International audience; OPA1 (Optic Atrophy 1) is a multi-isoform dynamin GTPase involved in the regulation of mitochondrial fusion and organization of the cristae structure of the mitochondrial inner membrane. Pathogenic OPA1 variants lead to a large spectrum of disorders associated with visual impairment due to optic nerve neuropathy. The aim of this study was to investigate the metabolomic consequences of complete OPA1 disruption in Opa1−/− mouse embryonic fibroblasts (MEFs) compared to their Opa1+/+ counterparts. Our non-targeted metabolomics approach revealed significant modifications of the concentration of several mitochondrial substrates, i.e. a decrease of aspartate, glutamate and α-ketoglutaric acid, and an increase of asparagine, glutamine and adenosine-5′-monophosphate, all related to aspartate metabolism. The signature further highlighted the altered metabolism of nucleotides and NAD together with deficient mitochondrial bioenergetics, reflected by the decrease of creatine/creatine phosphate and pantothenic acid, and the increase in pyruvate and glutathione. Interestingly, we recently reported significant variations of five of these molecules, including aspartate and glutamate, in the plasma of individuals carrying pathogenic OPA1 variants. Our findings show that the disruption of OPA1 leads to a remodelling of bioenergetic pathways with the central role being played by aspartate and related metabolites.

Comment on :Mutations in DNM1L, as in OPA1, result in dominant optic atrophy despite opposite effects on mitochondrial fusion and fission. [Brain. 2017]The expanding neurological phenotype of DNM1L-related disorders. [Brain. 2018]; International audience; Letter to the editor

International audience; AbstractBackrgroundEvaluation of the efficacy of oral cyclosporine A as a prophylactic agent in preventing second-eye involvement in Leber’s hereditary optic neuropathy (LHON) in a prospective, open-label, non-randomized, multicenter pilot study. Only LHON patients aged 18 years or more, with confirmed primary mitochondrial DNA mutations and strictly unilateral optic neuropathy occurring within 6 months prior to enrolment, were included in the study. All these patients, receiving treatment with oral cyclosporine (Neoral®, Novartis) at 2.5 mg/kg/day, were examined at three-month intervals for a year. The primary endpoint was the best corrected visual acuity in the unaffected eye; the secondary endpoints were the best corrected visual acuity in the first eye affected, the mean visual field defect on automated perimetry, the thickness of the perifoveal retinal ganglion cell inner plexiform layer, and the thickness of the peripapillary retinal nerve fiber layer in both eyes.ResultsAmong the 24 patients referred to our institution with genetically confirmed LHON, between July 2011 and April 2014, only five patients, four males and one female, fulfilled the inclusion criteria. Age at enrolment ranged from 19 to 42 years (mean: 27.2 years; median: 26 years), four patients harbored the m.11778G > A pathogenic variant, and one the m.14484 T > C pathogenic variant. The time-interval between the onset of symptoms and inclusion in the study ranged from 7 to 17 weeks (mean: 11.8 weeks; median: 9 weeks). Despite treatment with oral cyclosporine A, all patients eventually experienced bilateral eye involvement, occurring within 11–65 weeks after the initiation of treatment. Over the study time period, the average best corrected visual acuity worsened in the first eye affected; by the end of the study, both eyes were equally affected.ConclusionsOral cyclosporine, at 2.5 mg/kg/day, did not prevent second-eye involvement in patients with strictly unilateral Leber’s hereditary optic neuropathy.Trial registrationClinicalTrials.gov Identifier: NCT02176733. Registrated June 25, 2014.

International audience; Purpose: To determine the plasma metabolomic signature of the exfoliative syndrome (XFS), the most common cause worldwide of secondary open-angle glaucoma.Methods: We performed a targeted metabolomic study, using the standardized p180 Biocrates Absolute IDQ p180 kit with a QTRAP 5500 mass spectrometer, to compare the metabolomic profiles of plasma from individuals with XFS (n = 16), and an age- and sex-matched control group with cataract (n = 18).Results: A total of 151 metabolites were detected correctly, 16 of which allowed for construction of an OPLS-DA model with a good predictive capability (Q2cum = 0.51) associated with a low risk of over-fitting (permQ2 = -0.48, CV-ANOVA P-value Conclusions: We identified a significant metabolomic signature in the plasma of individuals with XFS. Paradoxically, this signature, characterized by lower concentrations of the neuroprotective spermine and spermidine polyamines than in controls, partially overlaps the plasma metabolomic profile associated with insulin resistance, despite the absence of evidence of insulin resistance in XFS.

International audience; Purpose: Dominant optic atrophy (DOA; MIM [Mendelian Inheritance in Man] 165500), resulting in retinal ganglion cell degeneration, is mainly caused by mutations in the optic atrophy 1 (OPA1) gene, which encodes a dynamin guanosine triphosphate (GTP)ase involved in mitochondrial membrane processing. This work aimed at determining whether plasma from OPA1 pathogenic variant carriers displays a specific metabolic signature.Methods: We applied a nontargeted clinical metabolomics pipeline based on ultra-high-pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) allowing the exploration of 500 polar metabolites in plasma. We compared the plasma metabolic profiles of 25 patients with various OPA1 pathogenic variants and phenotypes to those of 20 healthy controls. Statistical analyses were performed using univariate and multivariate (principal component analysis [PCA], orthogonal partial least-squares discriminant analysis [OPLS-DA]) methods and a machine learning approach, the Biosigner algorithm.Results: A robust and relevant predictive model characterizing OPA1 individuals was obtained, based on a complex panel of metabolites with altered concentrations. An impairment of the purine metabolism, including significant differences in xanthine, hypoxanthine, and inosine concentrations, was at the foreground of this signature. In addition, the signature was characterized by differences in urocanate, choline, phosphocholine, glycerate, 1-oleoyl-rac-glycerol, rac-glycerol-1-myristate, aspartate, glutamate, and cystine concentrations.Conclusions: This first metabolic signature reported in the plasma of patient carrying OPA1 pathogenic variants highlights the unexpected involvement of purine metabolism in the pathophysiology of DOA.

Background Inherited optic neuropathy has been ascribed to mutations in mitochondrial fusion/fission dynamics genes, nuclear and mitochondrial DNA-encoded respiratory enzyme genes or nuclear genes of poorly known mitochondrial function . However, the disease causing gene remains unknown in many families. Methods We used exome sequencing in order to identify the gene responsible for isolated or syndromic optic atrophy in five patients from three independent families. Results We found homozygous or compound heterozygous missense and frameshift mutations in the gene encoding mitochondrial aconitase (ACO2 ) , a tricarboxylic acid cycle enzyme, catalysing interconversion of citrate into isocitrate. Unlike wild type ACO2, all mutant ACO2 proteins failed to complement the respiratory growth of a yeast aco1- deletion strain. Retrospective studies using patient-derived cultured skin fibroblasts revealed various degrees of deficiency in ACO2 activity, but also in ACO1 cytosolic activity. Conclusions Our study shows that autosomal recessive ACO2 mutations can cause either isolated or syndromic optic neuropathy. This observation identifies ACO2 as the second gene responsible for non-syndromic autosomal recessive optic neuropathies and provides evidence for a genetic overlap between isolated and syndromic forms, giving further support to the view that optic atrophy is a hallmark of defective mitochondrial energy supply.

International audience; Purpose:Dominant optic atrophy (MIM No. 165500) is a blinding condition related to mutations in OPA1, a gene encoding a large GTPase involved in mitochondrial inner membrane dynamics. Although several mouse models mimicking the disease have been developed, the pathophysiological mechanisms responsible for retinal ganglion cell degeneration remain poorly understood.Methods:Using a targeted metabolomic approach, we measured the concentrations of 188 metabolites in nine tissues, that is, brain, three types of skeletal muscle, heart, liver, retina, optic nerve, and plasma in symptomatic 11-month-old Opa1delTTAG/+ mice.Results:Significant metabolic signatures were found only in the optic nerve and plasma of female mice. The optic nerve signature was characterized by altered concentrations of phospholipids, amino acids, acylcarnitines, and carnosine, whereas the plasma signature showed decreased concentrations of amino acids and sarcosine associated with increased concentrations of several phospholipids. In contrast, the investigation of 3-month-old presymptomatic Opa1delTTAG/+ mice showed no specific plasma signature but revealed a significant optic nerve signature in both sexes, although with a sex effect. The Opa1delTTAG/+ versus wild-type optic nerve signature was characterized by the decreased concentrations of 10 sphingomyelins and 10 lysophosphatidylcholines, suggestive of myelin sheath alteration, and by alteration in the concentrations of metabolites involved in neuroprotection, such as dimethylarginine, carnitine, spermine, spermidine, carnosine, and glutamate, suggesting a concomitant axonal metabolic dysfunction.Conclusions:Our comprehensive metabolomic investigations revealed in symptomatic as well as in presymptomatic Opa1delTTAG/+ mice, a specific sensitiveness of the optic nerve to Opa1 insufficiency, opening new routes for protective therapeutic strategies.