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A syndrome of hypogonadism, alopecia, diabetes mellitus, mental retardation, deafness, and ECG abnormalities. GLO:1WM/01aug21:ana26096-fig-0001.jpg PHOTO (COLOR): . gl Targeted genetic testing of DCAF17 gene was undertaken since the clinical findings were suggestive of a Woodhouse-Sakati syndrome (WSS).1 The patient, born to consanguineous parents, is homozygous for the c.436delC variation in DCAF17 gene. Mutations in C2orf37, encoding a nucleolar protein, cause hypogonadism, alopecia, diabetes mellitus, mental retardation, and extrapyramidal syndrome. [Extracted from the article]

Abstract: Mitochondrial respiratory chain deficiencies represent one of the major causes of metabolic disorders that are related to genetic defects in mitochondrial or nuclear DNA. The mitochondrial protein synthesis allows the synthesis of the 13 respiratory chain subunits encoded by mtDNA. Altogether, about 100 different proteins are involved in the translation of the 13 proteins encoded by the mitochondrial genome emphasizing the considerable investment required to maintain mitochondrial genetic system. Mitochondrial protein synthesis deficiency can be caused by mutations in any component of the translation apparatus including tRNA, rRNA and proteins. Mutations in mitochondrial rRNA and tRNAs have been first identified in various forms of mitochondrial disorders. Moreover abnormal translation due to mutation in nuclear genes encoding tRNA-modifying enzymes, ribosomal proteins, aminoacyl-tRNA synthetases, elongation and termination factors and translational activators have been successively described. These deficiencies are characterized by a huge clinical and genetic heterogeneity hampering to establish genotype–phenotype correlations and an easy diagnosis. One can hypothesize that a new technique for gene identification, such as exome sequencing will rapidly allow to expand the list of genes involved in abnormal mitochondrial protein synthesis. [Copyright &y& Elsevier]

Abstract: Mitochondrial DNA (mtDNA) depletion is characterized by a profound reduction of mtDNA copy number. The maintenance of mtDNA copy number requires several nuclear-encoded factors involved in replication and in dNTP supply. In the past decade mutations in several of these factors have been reported in a growing number of syndromes. This article reviews the current knowledge of genes causing mitochondrial DNA depletion syndromes. [Copyright &y& Elsevier]

Abstract: The mitochondrial respiratory chain (RC) results from the expression of both mitochondrial and nuclear genes. The number of disease-causing mutations in nuclear genes is steadily growing and mitochondrial DNA (mtDNA) deletions and mutations account for no more than 15–20% of pediatric patients. Unfortunately, the disease-causing mutations have been identified for only a small number of patients. Thus, elucidating the genetic bases of RC is both essential for genetic diagnosis of patients and for fundamental knowledge of these disorders. The molecular diagnostics of mitochondrial disorders come under both genetic diagnosis and research. Indeed, identification of a new gene in a specific patient allows to perform genetic diagnosis in other families and identification of mutations in already known disease-causing genes allows to constitute a cohort of patients for further functional studies. Thus, elucidating the genetic bases of RC deficiency is an essential task that needs the use of several appropriate strategies. Fine phenotypage of patients and candidate gene screening is the first step for the constitution of a well-characterized cohort of patients. Genetic mapping has to be used in large families. This approach is greatly enhanced in the case of consanguineous families. The consanguinity of the parents should also lead to test genetic markers surrounding the gene loci rather than to directly sequence several candidate genes. However, the main problem is encountered in the cases of sporadic cases for which no genetic approaches can be developed. In these cases, functional complementation by human chromosomes or cDNA is the only presently available strategy. [Copyright &y& Elsevier]

Friedreich''s ataxia (FRDA) is an autosomal recessive neurodegenerative disease causing limb and gait ataxia and cardiomyopathy. The disease gene encodes a mitochondrial protein of unknown function, frataxin. The loss of functional frataxin is caused by a large GAA trinucleotide expansion in the first intron of the gene, thus impairing gene transcription. The lack of frataxin appears to result primarily in disabled recruitment of early antioxidant defenses, resulting in oxidative insult to the highly sensitive iron-sulfur proteins aconitase and three mitochondrial respiratory chain complexes (I–III). Accordingly, antioxidant-based therapy appears promising in counteracting the course of the disease. [ABSTRACT FROM AUTHOR]

The oxidative stress possibly resulting from an inherited respiratory chain (RC) deficiency was investigated in a series of human cultured skin fibroblasts presenting either ubiquinone depletion or isolated defect of the various RC complexes. Taken as an index for superoxide overproduction, a significant induction of superoxide dismutase activity was observed in complex V-deficient fibroblasts harboring the NARP-mutation in the ATPase 6 gene. Superoxide dismutase induction was also noticed, albeit to a lesser extent, in complex II-deficient fibroblasts with a mutation in the nuclear gene encoding the flavoprotein subunit of the succinate dehydrogenase. No sign of oxidative stress could be found in ubiquinone-depleted fibroblasts. In all cases but complex IV-defect, increased oxidative stress was associated with increased cell death. In glucose-rich medium, apoptosis appeared as the main cell death process associated with all types of RC defect. However, similar to the great variations in oxidative stress associated with the various types of RC defect, we found that apoptotic features differed noticeably between defects. No indication of increased cell death was found in ubiquinone-depleted fibroblasts. [ABSTRACT FROM AUTHOR]

The succinate dehydrogenase consists of only four subunits, all nuclearly encoded, and is part of both the respiratory chain and the Krebs cycle. Mutations in the four genes encoding the subunits of the mitochondrial respiratory chain succinate dehydrogenase have been recently reported in human and shown to be associated with a wide spectrum of clinical presentations. Although a comparatively rare deficiency in human, molecularly defined succinate dehydrogenase deficiency has already been found to cause encephalomyopathy in childhood, optic atrophy or tumor in adulthood. Because none of the typical housekeeping genes encoding this respiratory chain complex is known to present tissue-specific isoforms, the tissue-specific involvement represents a quite intriguing question, which is mostly addressed in this review. A differential impairment of electron flow through the respiratory chain, handling of oxygen, and/or metabolic blockade possibly associated with defects in the different subunits that can be advocated to account for tissue-specific involvement is discussed. [Copyright &y& Elsevier]

A new method for the continuous assay of oxaloacetate released or taken up by plant mitochondria during malate oxidation is described. It is based on the continuous spectrophotometric recording of the reduction level of externally added NAD+ (0.4 mM) to a mitochondrial preparation. In the presence of 20 mM malate and of externally added malate dehydrogenase (EC 1.1.1.37), an equilibrium is reached in- stantaneously, bringing about a partial reduction of NAD+ and the production of a proportional amount of oxaloacetate (OAA). Owing to the presence of a very active OAA carrier on the inner mitochondrial membrane, the concentration at the equilibrium position of the reactants of the external MDH is permanently displaced by the OAA released or taken up by the mitochandria. Therefore, changes in OAA concentration can be followed from the measurement of the reduction level of the external NAD+. This method appears as sensitive as the classical enzymatic method, but is much more rapid and requires much less mitochondrial protein. The proposed method was applied to Percoll-purified mitochondria from the leaves of a CAM plant, Kalanchoë blossftldiana Poelln. cv. Tom Thumb. The simultaneous recording of the change in OAA concentration and of oxygen uptake during malate oxidation emphasizes the major control exerted by OAA on the rate of malate oxidation. [ABSTRACT FROM AUTHOR]

Friedreich's ataxia (FRDA) is a neuro-degenerative disease causing limb and gait ataxia and hypertrophic cardiomyopathy. It results from a triplet expansion in the first intron of the frataxin gene encoding a mitochondrial protein of yet unknown function. Cells with low frataxin content display generalized deficiency of mitochondrial iron-sulfur cluster-containing proteins, which presumably denotes overproduction of superoxide radicals in these organelles. Idebenone, a short-chain quinone, may act as a potent free radical scavenger protecting mitochondria against oxidative stress. We therefore carried out an open trial of idebenone (oral supplementation; 5 mg/kg/day) in a large series of FRDA patients and followed their left ventricular mass and function. Consistent and definitive worsening being observed in the natural course of the disease and cardiac hypertrophy having no chance of spontaneous reversal and to be subject to a placebo effect, the patient's heart status before and after the treatment was used to unambiguously establish the effect of the drug. After six months, heart ultrasound revealed more than 20% reduction of left ventricular mass in about half of the patients (p<0.001) and no significant change in the other half. Since any measurable reversion of this pathogenic trait is highly significant, this demonstrates the efficiency of idebenone in controlling heart hypertrophy in FRDA. Owing to the absence of side effects of the drug, idebenone (up to 15 mg/kg/day) should be prescribed for FRDA patients continuously as early as possible. [ABSTRACT FROM AUTHOR]

Ubiquinone (named because of its ubiquitous presence in organisms) functions as an electron carrier in the mitochondrial respiratory chain. have described the presence of new actors in the biosynthesis of this venerable molecule. [Copyright &y& Elsevier]

Genetic defects of mitochondrial energy supply can give rise to a variety of symptoms and virtually any organ or tissue can be involved. In particular, cardiomyopathy can be the presenting symptom of a respiratory enzyme deficiency in infancy. Alternatively, cardiomyopathy frequently occurs in the course of these diseases. Multi-organ involvement is usually regarded as a contraindication for heart transplantation in metabolic disorders. Yet, since the clinical expression of respiratory enzyme deficiency can be limited to the myocardium, it is reasonable to consider heart transplantation in mitochondrial cardiomyopathy.

STUDY QUESTION Does mitochondrial deficiency affect human embryonic preimplantation development? SUMMARY ANSWER The presence of a pathogenic mitochondrial variant triggers changes in the gene expression of preimplantation human embryos, compromising their development, cell differentiation, and survival. WHAT IS KNOWN ALREADY Quantitative and qualitative anomalies of mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain unexplained. STUDY DESIGN, SIZE, DURATION Taking advantage of the preimplantation genetic testing for mitochondrial disorders in at-risk couples, we have compared gene expression of 9 human embryos carrying pathogenic variants in either mtDNA genes or nuclear genes encoding mitochondrial protein to 33 age-matched control embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS Single-embryo transcriptomic analysis was performed on whole human blastocyst embryos donated to research. MAIN RESULTS AND THE ROLE OF CHANCE Specific pathogenic mitochondrial variants downregulate gene expression in preimplantation human embryos [566 genes in oxidative phosphorylation (OXPHOS)-deficient embryos], impacting transcriptional regulators, differentiation factors, and nuclear genes encoding mitochondrial proteins. These changes in gene expression primarily alter OXPHOS and cell survival pathways. LIMITATIONS, REASONS FOR CAUTION The number of OXPHOS-deficient embryos available for the study was limited owing to the rarity of this material. However, the molecular signature shared by all these embryos supports the relevance of the findings. WIDER IMPLICATIONS OF THE FINDINGS While identification of reliable markers of normal embryonic development is urgently needed in ART, our study prompts us to consider under-expression of the targeted genes reported here, as predictive biomarkers of mitochondrial dysfunction during preimplantation development. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by the 'Association Française contre les Myopathies (AFM-Téléthon)' and the 'La Fondation Maladies Rares'. No competing interests to declare. TRIAL REGISTRATION NUMBER N/A. [ABSTRACT FROM AUTHOR]

Abstract: Coenzyme Q10 (CoQ10) plays a pivotal role in oxidative phosphorylation (OXPHOS) as it distributes electrons between the various dehydrogenases and the cytochrome segments of the respiratory chain. Primary coenzyme Q10 deficiency is a rare, but possibly treatable, autosomal recessive condition with four major clinical presentations, an encephalomyopathic form, a generalized infantile variant with severe encephalopathy and renal disease, a myopathic form and an ataxic form. The diagnosis of ubiquinone deficiency is supported by respiratory chain analysis and eventually by the quantification of CoQ10 in patient tissues. We review here the infantile and pediatric quinone deficiency diseases as well as the clinical improvement after oral CoQ10 therapy. The clinical heterogeneity of ubiquinone deficiency is suggestive of a genetic heterogeneity that should be related to the large number of enzymes, and corresponding genes, involved in ubiquinone biosynthesis. [Copyright &y& Elsevier]

Transcription of mitochondrial DNA generates long polycistronic precursors whose nucleolytic cleavage yields the individual mtDNA-encoded transcripts. In most cases, this cleavage occurs at the 5′- and 3′-ends of tRNA sequences by the concerted action of RNAseP and RNaseZ/ELAC2 endonucleases, respectively. Variants in the ELAC2 gene have been predominantly linked to severe to mild cardiomyopathy that, in its milder forms, is accompanied by variably severe neurological presentations. Here, we report five patients from three unrelated families. Four of the patients presented mild to moderate cardiomyopathy and one died at 1 year of age, one patient had no evidence of cardiomyopathy. The patients had variable neurological presentations that included intellectual disability, ataxia, refractory epilepsy, neuropathy and deafness. All patients carried previously unreported missense and nonsense variants. Enzymatic analyses showed multiple OXPHOS deficiencies in biopsies from two patients, whereas immunoblot analyses revealed a decreased abundance of ELAC2 in fibroblasts from three patients. Northern blot analysis revealed an accumulation of unprocessed mt-tRNAVal-precursor consistent with the role of ELAC2 in transcript processing. Our study expands the genetic spectrum of ELAC2-linked disease and suggests that cardiomyopathy is not an invariably present clinical hallmark of this pathology. [ABSTRACT FROM AUTHOR]

Abstract: Out of a series of 30 French patients with Pearson syndrome, we report on two patients with an atypical presentation, which include growth deficiency, pancytopaenia, tubulopathy and absence of exocrine pancreas dysfunction. Patient 1, a 4-year-old boy with a past history of pancytopaenia and transient metabolic acidosis at 13 months of age, presented at 2½ years of age with severe tubulopathy of de Toni–Debré–Fanconi type, growth retardation, metabolic lactic acidosis and mild cytolysis. Despite normal exocrine pancreatic function, study of mitochondrial DNA revealed a 3.5kb deletion. Patient 2 had a personal history of pancytopaenia requiring blood transfusions at 11 months of age and presented with severe intractable proximal and distal tubulopathy at 2 years of age. Exocrine pancreatic deficiency could not be evidenced and post-mortem studies revealed a 4.9kb deletion of the mitochondrial DNA. A review of the literature revealed three patients presenting with Pearson syndrome and tubulopathy with normal pancreatic function and highlights delay in diagnosis in those three patients. The series of 30 French patients with Pearson syndrome also revealed that tubulopathy was present in 7/30 cases (23%), with variable outcome. In conclusion, Pearson syndrome should be screened for in children presenting with the association of growth retardation, anaemia/pancytopaenia, lactic acidosis and tubulopathy, even in the absence of exocrine pancreatic deficiency. [Copyright &y& Elsevier]

Most mitochondrial proteins are synthesized in the cytosol and targeted to mitochondria via N-terminal mitochondrial targeting signals (MTS) that are proteolytically removed upon import. Sometimes, MTS removal is followed by a cleavage of an octapeptide by the mitochondrial intermediate peptidase (MIP), encoded by the MIPEP gene. Previously, MIPEP variants were linked to four cases of multisystemic disorder presenting with cardiomyopathy, developmental delay, hypotonia and infantile lethality. We report here a patient carrying compound heterozygous MIPEP variants—one was not previously linked to mitochondrial disease—who did not have cardiomyopathy and who is alive at the age of 20 years. This patient had developmental delay, global hypotonia, mild optic neuropathy and mild ataxia. Functional characterization of patient fibroblasts and HEK293FT cells carrying MIPEP hypomorphic alleles demonstrated that deficient MIP activity was linked to impaired post-import processing of subunits from four of the five OXPHOS complexes and decreased abundance and activity of some of these complexes in human cells possibly underlying the development of mitochondrial disease. Thus, our work expands the genetic and clinical spectrum of MIPEP -linked disease and establishes MIP as an important regulator of OXPHOS biogenesis and function in human cells. • MIPEP variants cause neurological disease in the absence of cardiomyopathy. • Subunits from four of the five OXPHOS complexes are MIP substrates. • OXA1L is a novel MIP substrate. • MIP-mediated processing is an important regulator of OXPHOS function. [ABSTRACT FROM AUTHOR]

Unlabelled: Based on the study of three unrelated families, we report what we believe to be the first in vivo evidence of muscle ATPase deficiency in individuals carrying the neurogenic weakness, ataxia and retinitis pigmentosa (NARP) syndrome mtDNA mutation (T8993G). Since plasma citrulline was consistently low in 4/5 patients, we suggest that the NARP mutation caused complex V deficiency in the small intestine as well, thus reducing the availability of mitochondrial ATP required for citrulline synthesis.Conclusion: We suggest giving consideration to hypocitrullinaemia as a hallmark of the neurogenic weakness, ataxia and retinitis pigmentosa syndrome mutation and more generally of impaired oxidative phosphorylation in the small intestine in vivo. [ABSTRACT FROM AUTHOR]

Patients with PS have a dismal prognosis, most patients die before the age of 5 years.3,4 The few patients with PS surviving into later childhood develop a Kearns-Sayre syndrome. 2 The blood count at initial BM examination is given except for patient PSR23 in whom BM examination was not performed; instead the blood count at time of genetic diagnosis of PS is provided. BM cellularity was studied in BM biopsy in eight patients (§), in the other patients, the cell content was evaluated in BM smears. Keywords: Pearson syndrome; mitochondrial disease; single large-scale mitochondrial DNA deletion; hematological recovery EN Pearson syndrome mitochondrial disease single large-scale mitochondrial DNA deletion hematological recovery 1283 1287 5 06/18/21 20210615 NES 210615 Pearson syndrome (PS), a rare multisystem mitochondrial disorder caused by single large-scale mitochondrial DNA (mtDNA) deletions (SLSMDs), commonly presents with anaemia in infancy.1,2 Progressive multi-organ dysfunction such as lactic acidosis, pancreatic and renal dysfunction, failure to thrive and endocrine disorders can develop during the course.1,3 Interestingly, anaemia often resolves spontaneously; the frequency and prognostic impact of this observation is unknown. [Extracted from the article]

Mitochondrial aminoacyl-tRNA synthetases—encoded by ARS2 genes—are evolutionarily conserved enzymes that catalyse the attachment of amino acids to their cognate tRNAs, ensuring the accuracy of the mitochondrial translation process. ARS2 gene mutations are associated with a wide range of clinical presentations affecting the CNS. Two senior neuroradiologists analysed brain MRI of 25 patients (age range: 3 d–25 yrs.; 11 males; 14 females) with biallelic pathogenic variants of 11 ARS2 genes in a retrospective study conducted between 2002 and 2019. Though several combinations of brain MRI anomalies were highly suggestive of specific aetiologies (DARS2 , EARS2 , AARS2 and RARS2 mutations), our study detected no MRI pattern common to all patients. Stroke-like lesions were associated with pathogenic SARS2 and FARS2 variants. We also report early onset cerebellar atrophy and calcifications in AARS2 mutations, early white matter involvement in RARS2 mutations, and absent involvement of thalami in EARS2 mutations. Finally, our findings show that normal brain MRI results do not exclude the presence of ARS2 mutations: 5 patients with normal MRI images were carriers of pathogenic IARS2 , YARS2 , and FARS2 variants. Our study extends the spectrum of brain MRI anomalies associated with pathogenic ARS2 variants and suggests ARS2 mutations are largely underdiagnosed. [ABSTRACT FROM AUTHOR]

Aim: To investigate cerebral blood flow (CBF) in acute episodes of Leigh syndrome compared with basal state in patients carrying pathogenic mitochondrial disease gene variants responsible for neurometabolic disorders. Method: Arterial spin labelling (ASL) magnetic resonance imaging (MRI) sequences were used to measure CBF in 27 patients with mitochondrial respiratory chain enzyme deficiencies, ascribed to pathogenic variants of reported disease genes who were undergoing either urgent neuroimaging for acute episodes of Leigh syndrome (Group I: 15 MRI, seven females, eight males; mean age 7y; range 7mo–14y) or routine brain MRI (Group II: 15 MRI, eight females, seven males; mean age 5y 2mo; range 2mo–12y). Results: Patients displayed markedly increased CBF in the striatum (2.8‐fold greater, p<0.001 [1.05–2.53]) during acute episodes of Leigh syndrome compared to basal conditions. Detection of elevated CBF preceded identification of structural MRI lesions in four out of 15 cases. Interpretation: Our results suggest that increased CBF is an overt hallmark of Leigh syndrome episodes and ASL MRI sequences should facilitate early diagnosis of acute episodes of Leigh syndrome, especially during the first attack in young children, when structural MRI is insufficiently informative. [ABSTRACT FROM AUTHOR]

• Direct evidence of diaphragmatic dysfunction in mitochondrial myopathies is reported. • Diaphragmatic dysfunction caused alveolar hypoventilation requiring noninvasive ventilation. • Weakness of the global inspiratory and expiratory muscles was also demonstrated. • Respiratory muscle weakness contrasted with preserved peripheral muscle strength. • Esogastric pressure measurements are useful to document diaphragmatic dysfunction. Diaphragmatic dysfunction has been reported in congenital myopathies, muscular dystrophies, and occasionally, mitochondrial respiratory chain deficiency. Using a minimally invasive procedure in 3 young girls, 1 with a heteroplasmic MT-CYB mutation and 2 with biallelic pathogenic TK2 variants, we provided functional evidence of diaphragmatic dysfunction with global respiratory muscle weakness in mitochondrial respiratory chain deficiency. Analysis of respiratory muscle performance using esogastric pressures revealed paradoxical breathing and severe global inspiratory and expiratory muscle weakness with a sniff esophageal inspiratory pressure and a gastric pressure during cough averaging 50% and 40% of predicted values, respectively. This diaphragmatic dysfunction was responsible for severe undiagnosed nocturnal hypoventilation, requiring noninvasive ventilation. Our results underline the interest of this minimally invasive procedure for the evaluation of respiratory muscle performance and its potential value for the monitoring of future clinical trials in respiratory chain deficiency. [ABSTRACT FROM AUTHOR]

Isolated complex III (CIII) deficiencies are among the least frequently diagnosed mitochondrial disorders. Clinical symptoms range from isolated myopathy to severe multi-systemic disorders with early death and disability. To date, we know of pathogenic variants in genes encoding five out of 10 subunits and five out of 13 assembly factors of CIII. Here we describe rare bi-allelic variants in the gene of a catalytic subunit of CIII, UQCRFS1, which encodes the Rieske iron-sulfur protein, in two unrelated individuals. Affected children presented with low CIII activity in fibroblasts, lactic acidosis, fetal bradycardia, hypertrophic cardiomyopathy, and alopecia totalis. Studies in proband-derived fibroblasts showed a deleterious effect of the variants on UQCRFS1 protein abundance, mitochondrial import, CIII assembly, and cellular respiration. Complementation studies via lentiviral transduction and overexpression of wild-type UQCRFS1 restored mitochondrial function and rescued the cellular phenotype, confirming UQCRFS1 variants as causative for CIII deficiency. We demonstrate that mutations in UQCRFS1 can cause mitochondrial disease, and our results thereby expand the clinical and mutational spectrum of CIII deficiencies. [ABSTRACT FROM AUTHOR]

Background Primary microcephaly and profound global developmental delay have been considered the core clinical phenotype in patients with bi-allelic PRUNE1 mutations. Methods Linkage analysis and whole-exome sequencing (WES) in a multiplex family and extraction of further cases from a WES repository containing 571 children with severe developmental disabilities and neurologic symptoms. Results We identified bi-allelic PRUNE1 mutations in twelve children from six unrelated families. All patients who survived beyond the first 6 months of life had early-onset global developmental delay, bilateral spastic paresis, dysphagia and difficult-to-treat seizures, while congenital or later-evolving microcephaly was not a consistent finding. Brain MRI showed variable anomalies with progressive cerebral and cerebellar atrophies and T2- hyperintense brain stem lesions. Peripheral neuropathy was documented in five cases. Disease course was progressive in all patients and eight children died in the first or early second decade of life. In addition to the previously reportedmissensemutation p. (Asp106Asn), we observed a novel homozygous missense variant p.(Leu172Pro) and a homozygous contiguous gene deletion encompassing most of the PRUNE1 gene and part of the neighboring BNIPL gene. Conclusions PRUNE1 deficiency causes severe early-onset disease affecting the central and peripheral nervous systems. Microcephaly is probably not a universal feature. [ABSTRACT FROM AUTHOR]

Human mitochondrial aminoacyl-tRNA synthetases (mtaaRSs) are key enzymes in the mitochondrial protein translation system and catalyze the charging of amino acids on their cognate tRNAs. Mutations in their nuclear genes are associated with pathologies having a broad spectrum of clinical phenotypes, but with no clear molecular mechanism(s). For example, mutations in the nuclear genes encoding mt-AspRS and mt-ArgRS are correlated with the moderate neurodegenerative disorder leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) and with the severe neurodevelopmental disorder pontocerebellar hypoplasia type 6 (PCH6), respectively. Previous studies have shown no or only minor impacts of these mutations on the canonical properties of these enzymes, indicating that the role of the mt-aaRSs in protein synthesis is mostly not affected by these mutations, but their effects on the mitochondrial localizations of aaRSs remain unclear. Here, we demonstrate that three human aaRSs, mt-AspRS, mt-ArgRS, and LysRS, each have a specific sub-mitochondrial distribution, with mt-ArgRS being exclusively localized in the membrane, LysRS exclusively in the soluble fraction, and mt-AspRS being present in both. Chemical treatments revealed that mt-AspRs is anchored in the mitochondrial membrane through electrostatic interactions, whereas mt-ArgRS uses hydrophobic interactions. Wealso report that novel mutations in mt-AspRS and mt-ArgRS genes from individuals with LBSL and PCH6, respectively, had no significant impact on the mitochondrial localizations of mt- AspRS and mt-ArgRS. The variable sub-mitochondrial locations for these three mt-aaRSs strongly suggest the existence of additional enzyme properties, requiring further investigation to unravel the mechanisms underlying the two neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Freidreich ataxia (FRDA) is the most common hereditary ataxia, nearly 98% of patients harbouring homozygous GAA expansions in intron 1 of the FXN gene (NM_000144.4). The remaining patients are compound heterozygous for an expansion and a point mutation or an exonic deletion. Molecular screening for FXN expansion is therefore focused on (GAA)n expansion analysis, commonly performed by triplet repeat primed PCR (PT-PCR). We report on an initial pitfall in the molecular characterization of a 15 year-old girl with Freidreich ataxia (FRDA) who carried a rare deletion in intron 1 of the FXN gene. Due to this deletion TP-PCR failed to amplify the GAA expansion. This exceptional configuration induced misinterpretation of the molecular defect in this patient, who was first reported as having no FXN expansion. NGS analysis of a panel of 212 genes involved in nuclear mitochondrial disorders further revealed an intragenic deletion encompassing exons 4–5 of the FXN gene. Modified TP-PCR analysis confirmed the presence of a classical (GAA)n expansion located in trans . This case points out the possible pitfalls in molecular diagnosis of FRDA in affected patients and their relatives: detection of the FXN expansion may be impaired by several non-pathological or pathological variants around the FXN (GAA)n repeat. We propose a new molecular strategy to accurately detect expansion by TP-PCR in FRDA patients. [ABSTRACT FROM AUTHOR]

Respiratory chain complex I deficiency is the most frequently identified biochemical defect in childhood mitochondrial diseases. Clinical symptoms range from fatal infantile lactic acidosis to Leigh syndrome and other encephalomyopathies or cardiomyopathies. To date, disease-causing variants in genes coding for 27 complex I subunits, including 7 mitochondrial DNA genes, and in 11 genes encoding complex I assembly factors have been reported. Here, we describe rare biallelic variants in NDUFB8 encoding a complex I accessory subunit revealed by whole-exome sequencing in two individuals from two families. Both presented with a progressive course of disease with encephalo(cardio)myopathic features including muscular hypotonia, cardiac hypertrophy, respiratory failure, failure to thrive, and developmental delay. Blood lactate was elevated. Neuroimaging disclosed progressive changes in the basal ganglia and either brain stem or internal capsule. Biochemical analyses showed an isolated decrease in complex I enzymatic activity in muscle and fibroblasts. Complementation studies by expression of wild-type NDUFB8 in cells from affected individuals restored mitochondrial function, confirming NDUFB8 variants as the cause of complex I deficiency. Hereby we establish NDUFB8 as a relevant gene in childhood-onset mitochondrial disease. [ABSTRACT FROM AUTHOR]

Background Alterations in GFER gene have been associated with progressive mitochondrial myopathy, congenital cataracts, hearing loss, developmental delay, lactic acidosis and respiratory chain deficiency in 3 siblings born to consanguineous Moroccan parents by homozygosity mapping and candidate gene approach (OMIM#613076). Next generation sequencing recently confirmed this association by the finding of compound heterozygous variants in 19-year-old girl with a strikingly similar phenotype, but this ultra-rare entity remains however unknown from most of the scientific community. Materials and Methods Whole exome sequencing was performed as part of a 'diagnostic odyssey' for suspected mitochondrial condition in 2 patients, presenting congenital cataracts, progressive encephalomyopathy and hypotrophy and detected unreported compound heterozygous variants in GFER. Results Thanks to an international data sharing, we found 2 additional patients carrying compound heterozygous variants in GFER. Reverse phenotyping confirmed the phenotypical similarities between the 4 patients. Together with the first literature reports, the review of these 8 cases from 4 unrelated families enables us to better describe this apparently homogeneous disorder, with the clinical and biological stigmata of mitochondrial disease. Conclusion This report highlights the clinical utility of whole exome sequencing and reverse phenotyping for the diagnosis of ultra-rare diseases and underlines the importance of a broad data sharing for accurate clinical delineation of previously unrecognized entities. [ABSTRACT FROM AUTHOR]

Mitochondrial complex I deficiency results in a plethora of often severe clinical phenotypes manifesting in early childhood. Here, we report on three complex-I-deficient adult subjects with relatively mild clinical symptoms, including isolated, progressive exercise-induced myalgia and exercise intolerance but with normal later development. Exome sequencing and targeted exome sequencing revealed compound-heterozygous mutations in TMEM126B , encoding a complex I assembly factor. Further biochemical analysis of subject fibroblasts revealed a severe complex I deficiency caused by defective assembly. Lentiviral complementation with the wild-type cDNA restored the complex I deficiency, demonstrating the pathogenic nature of these mutations. Further complexome analysis of one subject indicated that the complex I assembly defect occurred during assembly of its membrane module. Our results show that TMEM126B defects can lead to complex I deficiencies and, interestingly, that symptoms can occur only after exercise. [ABSTRACT FROM AUTHOR]

Mitochondrial disorders are clinically and genetically diverse, with mutations in mitochondrial or nuclear genes able to cause defects in mitochondrial gene expression. Recently, mutations in several genes encoding factors involved in mt-tRNA processing have been identified to cause mitochondrial disease. Using whole-exome sequencing, we identified mutations in TRMT10C (encoding the mitochondrial RNase P protein 1 [MRPP1]) in two unrelated individuals who presented at birth with lactic acidosis, hypotonia, feeding difficulties, and deafness. Both individuals died at 5 months after respiratory failure. MRPP1, along with MRPP2 and MRPP3, form the mitochondrial ribonuclease P (mt-RNase P) complex that cleaves the 5′ ends of mt-tRNAs from polycistronic precursor transcripts. Additionally, a stable complex of MRPP1 and MRPP2 has m 1 R9 methyltransferase activity, which methylates mt-tRNAs at position 9 and is vital for folding mt-tRNAs into their correct tertiary structures. Analyses of fibroblasts from affected individuals harboring TRMT10C missense variants revealed decreased protein levels of MRPP1 and an increase in mt-RNA precursors indicative of impaired mt-RNA processing and defective mitochondrial protein synthesis. The pathogenicity of the detected variants—compound heterozygous c.542G>T (p.Arg181Leu) and c.814A>G (p.Thr272Ala) changes in subject 1 and a homozygous c.542G>T (p.Arg181Leu) variant in subject 2—was validated by the functional rescue of mt-RNA processing and mitochondrial protein synthesis defects after lentiviral transduction of wild-type TRMT10C . Our study suggests that these variants affect MRPP1 protein stability and mt-tRNA processing without affecting m 1 R9 methyltransferase activity, identifying mutations in TRMT10C as a cause of mitochondrial disease and highlighting the importance of RNA processing for correct mitochondrial function. [ABSTRACT FROM AUTHOR]

A 41-day-old infant who had severe metabolic acidosis, anemia, bleeding, hypoglycemia, and proximal tubulopathy was diagnosed with Pearson syndrome. Fibrosis in the liver, severe iron deposition in hepatocytes, and multiple renal cortical cysts were found on postmortem examination. Southern blot analysis of mitochondrial DNA obtained from peripheral blood revealed a heteroplasmic deletion of approximately 3.5 kilobases. [ABSTRACT FROM AUTHOR]

Background: Sengers syndrome is an autosomal recessive condition characterized by congenital cataract, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. Mutations in the acylglycerol kinase (AGK) gene have been recently described as the cause of Sengers syndrome in nine families. Methods: We investigated the clinical and molecular features of Sengers syndrome in seven new families; five families with the severe and two with the milder form. Results: Sequence analysis of AGK revealed compound heterozygous or homozygous predicted loss-of-function mutations in all affected individuals. A total of eight different disease alleles were identified, of which six were novel, homozygous c.523_524delAT (p.Ile175Tyrfs*2), c.424-1G > A (splice site), c.409C > T (p.Arg137*) and c.877 + 3G > T (splice site), and compound heterozygous c.871C > T (p.Gln291*) and c.1035dup (p.Ile346Tyrfs*39). All patients displayed perinatal or early-onset cardiomyopathy and cataract, clinical features pathognomonic for Sengers syndrome. Other common findings included blood lactic acidosis and tachydyspnoea while nystagmus, eosinophilia and cervical meningocele were documented in only either one or two cases. Deficiency of the adenine nucleotide translocator was found in heart and skeletal muscle biopsies from two patients associated with respiratory chain complex I deficiency. In contrast to previous findings, mitochondrial DNA content was normal in both tissues. Conclusion: We compare our findings to those in 21 previously reported AGK mutation-positive Sengers patients, confirming that Sengers syndrome is a clinically recognisable disorder of mitochondrial energy metabolism. [ABSTRACT FROM AUTHOR]

Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Presently, no efficient therapeutic treatment has been developed against this class of pathologies. Because most of deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mitochondrial DNA (mtDNA) coexist in the same cell, the shift in proportion between mutant and wild type molecules could restore mitochondrial functions. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and thus impact heteroplasmy level in cells bearing a large deletion in mtDNA. Here, we show that this strategy can be also applied to point mutations in mtDNA. We demonstrate that specifically designed RNA molecules containing structural determinants for mitochondrial import and 20-nucleotide sequence corresponding to the mutated region of mtDNA, are able to anneal selectively to the mutated mitochondrial genomes. After being imported into mitochondria of living human cells in culture, these RNA induced a decrease of the proportion of mtDNA molecules bearing a pathogenic point mutation in the mtDNA ND5 gene. [ABSTRACT FROM AUTHOR]

Abstract: Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and are associated with a wide range of clinical symptoms. We report a subject, born to consanguineous parents, with growth retardation and neurological deterioration. Multiple respiratory chain deficiency was found in muscle and fibroblasts of the subject as well as abnormal assembly of complexes I and IV. A microsatellite genotyping of the family members detected only one region of homozygosity on chromosome 17q24.2–q25.3 in which we focused our attention to genes involved in mitochondrial translation. We sequenced MRPL12, encoding the mitochondrial ribosomal protein L12 and identified a c.542C>T transition in exon 5 changing a highly conserved alanine into a valine (p.Ala181Val). This mutation resulted in a decreased steady-state level of MRPL12 protein, with altered integration into the large ribosomal subunit. Moreover, an overall mitochondrial translation defect was observed in the subject's fibroblasts with a significant reduction of synthesis of COXI, COXII and COXIII subunits. Modeling of MRPL12 shows Ala181 positioned in a helix potentially involved in an interface of interaction suggesting that the p.Ala181Val change might be predicted to alter interactions with the elongation factors. These results contrast with the eubacterial orthologues of human MRPL12, where L7/L12 proteins do not appear to have a selective effect on translation. Therefore, analysis of the mutated version found in the subject presented here suggests that the mammalian protein does not function in an entirely analogous manner to the eubacterial L7/L12 equivalent. [Copyright &y& Elsevier]

We report two children, born from consanguineous parents, who presented with early-onset refractory epilepsy associated with psychomotor delay, failure to thrive, blindness and deafness. Polarographic and spectrophotometric analyses in fibroblasts and liver revealed a respiratory chain (RC) dysfunction. Surprisingly, we identified a homozygous nonsense mutation in the GM3 synthase gene by using exome sequencing. GM3 synthase catalyzes the formation of GM3 ganglioside from lactosylceramide, which is the first step in the synthesis of complex ganglioside species. Mass spectrometry analysis revealed that the complete absence of GM3 ganglioside and its biosynthetic derivatives was associated with an upregulation of the alternative globoside pathway in fibroblasts. The accumulation of Gb3 and Gb4 globosides likely has a role in RC dysfunction and in the decrease of mitochondrial membrane potential leading to apoptosis, which we observed in fibroblasts. We show for the first time that GM3 synthase deficiency, responsible for early-onset epilepsy syndrome, leads to a secondary RC dysfunction. Our study highlights the role of secondary mitochondrial disorders that can interfere with the diagnosis and the evolution of other metabolic diseases. [ABSTRACT FROM AUTHOR]

Abstract: A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample. [Copyright &y& Elsevier]

Abstract: We report maternal uniparental disomy of chromosome 2 (matUPD2) in a 9-month-old girl presenting with hepatocerebral mitochondrial DNA depletion syndrome. This patient was homozygous for the c.352C>T (p.Arg118Cys) mutation in DGUOK gene. The proband''s mother was heterozygous for the mutation was absent in DNA of the father. For proband, the absence of paternal contribution at the DGUOK locus prompted us to exclude intragenic DGUOK deletion of the paternal allele with Multiplex ligation-dependent probe amplification (MLPA) analysis. We also excluded non-paternity by studying various markers at different loci. Then we performed an analysis of copy number variations and absence of heterozygosity (AOH) on the proband DNA using high resolution oligonucleotides microarray. Several large regions of AOH with no copy number change were detected on chromosome 2 and one of these AOH regions encompassed DGUOK gene. These results were confirmed with haplotype analysis using polymorphic markers. Informative SNPs and microsatellites markers spanning the whole chromosome 2 showed a matUPD2 with heterodisomy and isodisomy regions, the absence of paternal allele and presence of two maternal alleles, with only one maternal allele on the region of DGUOK locus in 2p13.1. This is the first demonstration of matUPD2 with segmental isodisomy at 2p13.1 locus in hepatocerebral mitochondrial DNA depletion syndrome. The identification of UPD2 will impact genetic counseling for the proband''s parents. Because the recurrence risk for UPD2 is very low, the risk for disease in further offspring for this couple is negligible. [Copyright &y& Elsevier]

Patients et Méthodes: Nous rapportons l’étude de 63 enfants (1991-2006) chez qui une maladie du foie a été l’occasion du diagnostic de mitochon-driopathie après exclusion des autres causes, un dysfonctionnement de l’un ou plusieurs des complexes enzymatiques de la chaîne respiratoire étant mis en évidence dans le foie (56 patients) ou un autre tissu (7pts). Résultats: Quatre modes de présentation ont été observés. Groupe I : insuffisance hépatocellulaire (IHC) néonatale (20 enfants) définie par un temps de Quick (TQ) < 40 % avant J8 ; on observe : ictère (19p), hépatomégalie (18p), atteinte neurologique (10p), déplétion de l’ADN mt (8/8), mutation du gène DGK (5/6) ; évolution vers la mort (16p dont 1 après TH)), ou vers l’amélioration de la biologie hépatique (4 p, recul 1-10 ans).Groupe II : IHC secondaire (21 p) définie par un TQ < 70 % entre 1 et 16 mois ; on observe : ictère (18p), hépatomégalie (16p), atteinte neurologique (10 p), déplétion de l’ADN mt (8/10), mutation du gène DGK (2/7) ; évolution vers la mort (8p, âge : 1.5m-20m), vers la nécessité d’une TH (9p, âge : 1.5 m-17m, 7 survivants) ou vers l’amélioration durable de la biologie hépatique (4 enfants ; recul 1-12 ans). Groupe III : cholestase avant 3 mois (13 pts) définie par la présence d’un ictère sans IHC avant 3 mois ; on observe : hépatomégalie (10p), atteinte neurologique (4p), déplétion de l’ADNmt (7/8) ; évolution vers l’IHC (6 p décédés dont 1 après TH et 4 atteintes neurologiques) ou vers l’amélioration de la biologie hépatique (7 p, recul 1-3 an s). Groupe IV : découverte fortuite (9p), avant 3 ans, d’une hépatomégalie (4p) ou d’une élévation des transaminases (5p) ; on observe : atteinte neurologique (3p), cirrhose (4 p), déplétion de l’ADNmt (3/4), mutation du gène POLG (2/2) ; évolution vers l’IHC (1p), vers une dégradation neurologique (3p) ou vers l’amélioration de la fonction hépatique (5 p). Conclusion: 1-une anomalie de la chaîne respiratoire mitochondriale peut se révéler chez le nouveau-né et le nourrisson par quatre présentations cliniques hépatiques ; 2-une déplétion de l’ADNmt est fréquemment présente ; 3-le pronostic est particulièrement grave dans les formes néonatales ; 4-une amélioration spontanée durable de la biologie hépatique est possible dans toutes les formes. [Copyright &y& Elsevier]

Multiple-respiratory-chain deficiency represents an important cause of mitochondrial disorders. Hitherto, however, mutations in genes involved in mtDNA maintenance and translation machinery only account for a fraction of cases. Exome sequencing in two siblings, born to consanguineous parents, with severe encephalomyopathy, choreoathetotic movements, and combined respiratory-chain defects allowed us to identify a homozygous PNPT1 missense mutation (c.1160A>G) that encodes the mitochondrial polynucleotide phosphorylase (PNPase). Blue-native polyacrylamide gel electrophoresis showed that no PNPase complex could be detected in subject fibroblasts, confirming that the substitution encoded by c.1160A>G disrupts the trimerization of the protein. PNPase is predominantly localized in the mitochondrial intermembrane space and is implicated in RNA targeting to human mitochondria. Mammalian mitochondria import several small noncoding nuclear RNAs (5S rRNA, MRP RNA, some tRNAs, and miRNAs). By RNA hybridization experiments, we observed a significant decrease in 5S rRNA and MRP-related RNA import into mitochondria in fibroblasts of affected subject 1. Moreover, we found a reproducible decrease in the rate of mitochondrial translation in her fibroblasts. Finally, overexpression of the wild-type PNPT1 cDNA in fibroblasts of subject 1 induced an increase in 5S rRNA import in mitochondria and rescued the mitochondrial-translation deficiency. In conclusion, we report here abnormal RNA import into mitochondria as a cause of respiratory-chain deficiency. [Copyright &y& Elsevier]

Abstract: MutS homologs play a central role in maintaining genetic stability. We show that MSH5 (MutS Homolog 5) is localized into the mitochondria of germ and somatic cells. This protein binds to mtDNA and interacts with the Twinkle helicase and the DNA polymerase gamma. hMSH5 stimulates mtDNA repair in response to DNA damage induced by oxidative stress. Furthermore, we observed a subsarcolemmal accumulation of hMSH5 in COX negative muscle fibers of patients presenting a mitochondrial myopathy. We report a novel localization for hMSH5 suggesting that this protein may have functions other than those known in meiotic recombination. [Copyright &y& Elsevier]

Abstract: Isolated complex I deficiency is a frequent cause of respiratory chain defects in childhood. In this study, we report our systematic approach with blue native PAGE (BN-PAGE) to study mitochondrial respiratory chain assembly in skin fibroblasts from patients with Leigh syndrome and CI deficiency. We describe five new NDUFS4 patients with a similar and constant abnormal BN-PAGE profile and present a meta-analysis of the literature. All NDUFS4 mutations that have been tested with BN-PAGE result in a constant and similar abnormal assembly profile with a complete loss of the fully assembled complex I usually due to a truncated protein and the loss of its canonical cAMP dependent protein kinase phosphorylation consensus site. We also report the association of abnormal brain MRI images with this characteristic BN-PAGE profile as the hallmarks of NDUFS4 mutations and the first founder NDUFS4 mutations in the North-African population. [Copyright &y& Elsevier]

Respiratory chain (RC) complexes are organized into supercomplexes forming 'respirasomes'. The mechanism underlying the interdependence of individual complexes is still unclear. Here, we show in human patient cells that the presence of a truncated COX1 subunit leads to destabilization of complex IV (CIV) and other RC complexes. Surprisingly, the truncated COX1 protein is integrated into subcomplexes, the holocomplex and even into supercomplexes, which however are all unstable. Depletion of the m-AAA protease AFG3L2 increases stability of the truncated COX1 and other mitochondrially encoded proteins, whereas overexpression of wild-type AFG3L2 decreases their stability. Both full-length and truncated COX1 proteins physically interact with AFG3L2. Expression of a dominant negative AFG3L2 variant also promotes stabilization of CIV proteins as well as the assembled complex and rescues the severe phenotype in heteroplasmic cells. Our data indicate that the mechanism underlying pathogenesis in these patients is the rapid clearance of unstable respiratory complexes by quality control pathways, rather than their impaired assembly. [ABSTRACT FROM AUTHOR]

Abstract: Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases. We report two novel GFM1 mutations in two unrelated patients with encephalopathy and liver failure respectively. The first patient had intrauterine growth retardation, seizures, encephalopathy and developmental delay. Brain MRI showed hypoplasia of the vermis and severe pontine atrophy of the brainstem that were similar to those reported in patients with mitochondrial translation deficiencies. The second patient had liver failure with hypoglycemia. Respiratory chain analysis showed a complex IV deficiency in muscle of both patients. A 10K SNP genotyping detected several regions of homozygosity in the two patients. In vitro translation deficiency prompted us to study genes involved in mitochondrial translation. Therefore, we sequenced the GFM1 gene, encoding the mitochondrial translation factor EFG1, included in a shared homozygous region and identified two different homozygous mutations (R671C and L398P). Modeling studies of EFG1 protein suggested that the R671C mutation disrupts an inter-subunit interface and could locally destabilize the mutant protein. The second mutation (L398P) disrupted the H-bond network in a rich-beta-sheet domain, and may have a dramatic effect on local structure. GFM1 mutations have been seldom reported and are associated with different clinical presentation. By modeling the structure of the protein and the position of the various mutations we suggest that the clinical phenotypes of the patients could be related to the localization of the mutations. [Copyright &y& Elsevier]

We investigated a family in which the index subject presented with severe congenital lactic acidosis and dysmorphic features associated with a cytochrome c oxidase (COX)-assembly defect and a specific decrease in the synthesis of COX I, the subunit that nucleates COX assembly. Using a combination of microcell-mediated chromosome transfer, homozygosity mapping, and transcript profiling, we mapped the gene defect to chromosome 12 and identified a homozygous missense mutation (c.88G>A) in C12orf62. C12orf62 was not detectable by immunoblot analysis in subject fibroblasts, and retroviral expression of the wild-type C12orf62 cDNA rescued the biochemical phenotype. Furthermore, siRNA-mediated knockdown of C12orf 62 recapitulated the biochemical defect in control cells and exacerbated it in subject cells. C12orf62 is apparently restricted to the vertebrate lineage. It codes for a very small (6 kDa), uncharacterized, single-transmembrane protein that localizes to mitochondria and elutes in a complex of ∼110 kDa by gel filtration. COX I, II, and IV coimmunoprecipated with an epitope-tagged version of C12orf62, and 2D blue-native-polyacrylamide-gel-electrophoresis analysis of newly synthesized mitochondrial COX subunits in subject fibroblasts showed that COX assembly was impaired and that the nascent enzyme complex was unstable. We conclude that C12orf62 is required for coordination of the early steps of COX assembly with the synthesis of COX I. [ABSTRACT FROM AUTHOR]

Background & Aims: Multiple respiratory chain deficiencies represent a common cause of mitochondrial diseases and often result in hepatic failure. A significant fraction of patients present mitochondrial DNA depletion but a number of cases remain unexplained. The aim of our study was to identify the disease causing gene in a kindred with intrauterine growth retardation, neonatal lactic acidosis, liver dysfunction and multiple respiratory chain deficiency in muscle. Methods: Homozygosity mapping was performed by 50K SNP genotyping and candidate genes were successively analyzed by direct sequencing on genomic DNA of the family members. Results: SNP genotyping detected several regions of homozygosity in which we focused our attention to genes involved in mitochondrial translation. We sequenced the TSFM gene, encoding the mitochondrial translation factor EFTs and identified a homozygous mutation changing a highly conserved arginine into a tryptophan (R312W). Conclusions: This mutation has been previously reported in two unrelated kindred presenting two distinct syndromes (fatal mitochondrial encephalomyopathy and hypertrophic cardiomyopathy respectively). The description of a third syndrome associated with a same TSFM mutation gives support to the broad clinical and genetic heterogeneity of mitochondrial translation deficiencies in human. It suggests that mitochondrial translation deficiency represents a growing cause of hepatic failure of mitochondrial origin in infants. [ABSTRACT FROM AUTHOR]

Chronic intestinal pseudo-obstruction (CIPO) is a severe disease of the digestive tract motility. In pediatric population, CIPO remains of unknown origin for most patients. Chronic intestinal pseudo-obstruction is also a common feature in the course of mitochondrial oxidative phosphorylation disorders related for some patients to mutations in TYMP, POLG1, mtDNA tRNA or tRNA genes. We hypothesized that CIPOs could be the presenting symptom of respiratory chain enzyme deficiency and thus we investigated oxidative phosphorylation in small bowel and/or colon smooth muscle of primary CIPO children. We studied eight children with CIPO and 12 pediatric controls. We collected clinical, radiological and pathological data and measured respiratory chain enzymatic activity in isolated smooth muscle of the small bowel and/or the colon. We also sequenced TYMP, POLG, mtDNA tRNA and tRNA genes. Neither pathological nor radiological data were in favor of a mitochondrial dysfunction. No respiratory chain enzyme deficiency was detected in CIPO children. In myogenic CIPO, respiratory enzymes and citrate synthase activities were increased in small bowel and/or colon whereas no abnormality was noted in neurogenic and unclassified CIPO. Levels of enzyme activities were higher in control small bowel than in control colon muscle. Sequencing of TYMP, POLG, mtDNA tRNA and tRNA genes and POLG gene did not reveal mutation for any of the patients. The normal enzymatic activities as the lack of radiological and genetic abnormalities indicate that, at variance with adult patients, oxidative phosphorylation deficiency is not a common cause of childhood CIPO. [ABSTRACT FROM AUTHOR]

Mitochondrial complex I deficiency is the most common oxidative phosphorylation defect. Mutations have been detected in mitochondrial and nuclear genes, but the genetics of many patients remain unresolved and new genes are probably involved. In a consanguineous family, patients presented easy fatigability, exercise intolerance and lactic acidosis in blood from early childhood. In muscle, subsarcolemmal mitochondrial proliferation and a severe complex I deficiency were observed. Exercise intolerance and complex I activity was improved by a supplement of riboflavin at high dosage. Homozygosity mapping revealed a candidate region on chromosome three containing six mitochondria-related genes. Four genes were screened for mutations and a homozygous substitution was identified in ACAD9 (c.1594C>T), changing the highly conserved arginine-532 into tryptophan. This mutation was absent in 188 ethnically matched controls. Protein modelling suggested a functional effect due to the loss of a stabilizing hydrogen bond in an α-helix and a local flexibility change. To test whether the ACAD9 mutation caused the complex I deficiency, we transduced fibroblasts of patients with wild-type and mutant ACAD9. Wild-type, but not mutant, ACAD9 restored complex I activity. An unrelated patient with the same phenotype was compound heterozygous for c.380G>A and c.1405C>T, changing arginine-127 into glutamine and arginine-469 into tryptophan, respectively. These amino acids were highly conserved and the substitutions were not present in controls, making them very probably pathogenic. Our data support a new function for ACAD9 in complex I function, making this gene an important new candidate for patients with complex I deficiency, which could be improved by riboflavin treatment. [ABSTRACT FROM PUBLISHER]

Despite advanced knowledge on the genetic basis of oxidative phosphorylation-related diseases, the molecular and/or cellular determinants for tissue-specific dysfunction are not completely understood. Here, we report the cellular events associated with mitochondrial respiratory Complex II deficiency occurring before cell death. Mutation or chronic inhibition of Complex II determined a large increase of basal and agonist-evoked Ca2+ signals in the cytosol and the mitochondria, in parallel with mitochondrial dysfunction characterized by membrane potential (Δψmit) loss, [ATP] reduction and increased reactive oxygen species production. Cytosolic and mitochondrial Ca2+ overload are linked to increased endoplasmic reticulum (ER) Ca2+ leakage, and to SERCA2b and PMCA proteasome-dependent degradation. Increased [Ca2+]mit is also contributed by decreased mitochondrial motility and increased ER-mitochondria contact sites. Interestingly, increased intracellular [Ca2+] activated on the one hand a compensatory Ca2+-dependent glycolytic ATP production and determined on the second hand mitochondrial pathology. These results revealed the primary function for Ca2+ signalling in the control of mitochondrial dysfunction and cellular bioenergetics outcomes linked to respiratory chain Complex II deficiency. [ABSTRACT FROM AUTHOR]