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1.
EMBO Rep ; 24(5): e56114, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-36929726

RESUMO

Vesicular transport is a means of communication. While cells can communicate with each other via secretion of extracellular vesicles, less is known regarding organelle-to organelle communication, particularly in the case of mitochondria. Mitochondria are responsible for the production of energy and for essential metabolic pathways in the cell, as well as fundamental processes such as apoptosis and aging. Here, we show that functional mitochondria isolated from Saccharomyces cerevisiae release vesicles, independent of the fission machinery. We isolate these mitochondrial-derived vesicles (MDVs) and find that they are relatively uniform in size, of about 100 nm, and carry selective protein cargo enriched for ATP synthase subunits. Remarkably, we further find that these MDVs harbor a functional ATP synthase complex. We demonstrate that these vesicles have a membrane potential, produce ATP, and seem to fuse with naive mitochondria. Our findings reveal a possible delivery mechanism of ATP-producing vesicles, which can potentially regenerate ATP-deficient mitochondria and may participate in organelle-to-organelle communication.


Assuntos
Mitocôndrias , Saccharomyces cerevisiae , Potenciais da Membrana , Mitocôndrias/metabolismo , Transporte Biológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo
2.
Genes Dev ; 30(17): 1991-2004, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27664238

RESUMO

Multiple transcriptional and epigenetic changes drive differentiation of embryonic stem cells (ESCs). This study unveils an additional level of gene expression regulation involving noncanonical, cap-independent translation of a select group of mRNAs. This is driven by death-associated protein 5 (DAP5/eIF4G2/NAT1), a translation initiation factor mediating IRES-dependent translation. We found that the DAP5 knockdown from human ESCs (hESCs) resulted in persistence of pluripotent gene expression, delayed induction of differentiation-associated genes in different cell lineages, and defective embryoid body formation. The latter involved improper cellular organization, lack of cavitation, and enhanced mislocalized apoptosis. RNA sequencing of polysome-associated mRNAs identified candidates with reduced translation efficiency in DAP5-depleted hESCs. These were enriched in mitochondrial proteins involved in oxidative respiration, a pathway essential for differentiation, the significance of which was confirmed by the aberrant mitochondrial morphology and decreased oxidative respiratory activity in DAP5 knockdown cells. Further analysis identified the chromatin modifier HMGN3 as a cap-independent DAP5 translation target whose knockdown resulted in defective differentiation. Thus, DAP5-mediated translation of a specific set of proteins is critical for the transition from pluripotency to differentiation, highlighting the importance of cap-independent translation in stem cell fate decisions.


Assuntos
Diferenciação Celular/genética , Fator de Iniciação Eucariótico 4G/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células-Tronco Embrionárias Humanas/citologia , Apoptose/genética , Corpos Embrioides/patologia , Fator de Iniciação Eucariótico 4G/genética , Técnicas de Silenciamento de Genes , Proteínas HMGN/genética , Proteínas HMGN/metabolismo , Humanos , Células-Tronco Pluripotentes/fisiologia
3.
J Inherit Metab Dis ; 46(2): 232-242, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36515074

RESUMO

Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.


Assuntos
Galactosemias , Humanos , Recém-Nascido , Galactosemias/diagnóstico , Triagem Neonatal/métodos , Projetos Piloto , UTP-Hexose-1-Fosfato Uridililtransferase , Racemases e Epimerases
4.
Brain ; 145(3): 872-878, 2022 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-34788402

RESUMO

Pathogenic variants in SOD1, encoding superoxide dismutase 1, are responsible for about 20% of all familial amyotrophic lateral sclerosis cases, through a gain-of-function mechanism. Recently, two reports showed that a specific homozygous SOD1 loss-of-function variant is associated with an infantile progressive motor-neurological syndrome. Exome sequencing followed by molecular studies, including cDNA analysis, SOD1 protein levels and enzymatic activity, and plasma neurofilament light chain levels, were undertaken in an infant with severe global developmental delay, axial hypotonia and limb spasticity. We identified a homozygous 3-bp in-frame deletion in SOD1. cDNA analysis predicted the loss of a single valine residue from a tandem pair (p.Val119/Val120) in the wild-type protein, yet expression levels and splicing were preserved. Analysis of SOD1 activity and protein levels in erythrocyte lysates showed essentially no enzymatic activity and undetectable SOD1 protein in the child, whereas the parents had ∼50% protein expression and activity relative to controls. Neurofilament light chain levels in plasma were elevated, implying ongoing axonal injury and neurodegeneration. Thus, we provide confirmatory evidence of a second biallelic variant in an infant with a severe neurological syndrome and suggest that the in-frame deletion causes instability and subsequent degeneration of SOD1. We highlight the importance of the valine residues at positions V119-120, and suggest possible implications for future therapeutics research.


Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/metabolismo , DNA Complementar , Humanos , Lactente , Mutação/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genética , Síndrome , Valina/genética
5.
Int J Mol Sci ; 23(8)2022 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-35456968

RESUMO

Cytochrome c oxidase (COX), a multimeric protein complex, is the final electron acceptor in the mitochondrial electron transfer chain. Primary COX deficiency, caused by mutations in either mitochondrial DNA or nuclear-encoded genes, is a heterogenous group of mitochondrial diseases with a wide range of presentations, ranging from fatal infantile to subtler. We previously reported a patient with primary COX deficiency due to a pathogenic variant in COX4I1 (encoding the common isoform of COX subunit 4, COX4-1), who presented with bone marrow failure, genomic instability, and short stature, mimicking Fanconi anemia (FA). In the present study, we demonstrated that accumulative DNA damage coincided primarily with proliferative cells in the patient's fibroblasts and in COX4i1 knockdown cells. Expression analysis implicated a reduction in DNA damage response pathways, which was verified by demonstrating impaired recovery from genotoxic insult and decreased DNA repair. The premature senescence of the COX4-1-deficient cells prevented us from undertaking additional studies; nevertheless, taken together, our results indicate replicative stress and impaired nuclear DNA damage response in COX4-1 deficiency. Interestingly, our in vitro findings recapitulated the patient's presentation and present status.


Assuntos
Deficiência de Citocromo-c Oxidase , Doenças Mitocondriais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Dano ao DNA , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos
6.
J Hum Genet ; 66(8): 835-840, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-33612823

RESUMO

MSTO1 is a cytoplasmic protein that modulates mitochondrial dynamics by promoting mitochondrial fusion. Mutations in the MSTO1 gene are responsible for an extremely rare condition characterized by early-onset myopathy and cerebellar ataxia. We report here two siblings from a large Ashkenazi Jewish family, presenting with a progressive neuromuscular disease characterized by ataxia and myopathy. By whole exome sequencing, we found a novel homozygous missense mutation (c.1403T>A, p.Leu468Gln) in MSTO1. Studies performed on fibroblasts from the index patient demonstrated the pathogenic role of the identified variant; we found that MSTO1 protein level was reduced and that mitochondrial network was fragmented or formed enlarged structures. Moreover, patient's cells showed reduced mitochondrial DNA amount. Our report confirms that MSTO1 mutations are typically recessive, and associated with clinical phenotypes characterized by early-onset muscle impairment and ataxia, often with upper motor neuron signs and varied cognitive impairment.


Assuntos
Ataxia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , DNA Mitocondrial/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Musculares/genética , Adulto , Feminino , Fibroblastos/metabolismo , Homozigoto , Humanos , Judeus/genética , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Irmãos , Sequenciamento do Exoma , Adulto Jovem
7.
J Inherit Metab Dis ; 44(3): 606-617, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33190319

RESUMO

Urea cycle disorders (UCDs), including OTC deficiency (OTCD), are life-threatening diseases with a broad clinical spectrum. Early diagnosis and initiation of treatment based on a newborn screening (NBS) test for OTCD with high specificity and sensitivity may contribute to reduction of the significant complications and high mortality. The efficacy of incorporating orotic acid determination into routine NBS was evaluated. Combined measurement of orotic acid and citrulline in archived dried blood spots from newborns with urea cycle disorders and normal controls was used to develop an algorithm for routine NBS for OTCD in Israel. Clinical information and genetic confirmation results were obtained from the follow-up care providers. About 1147986 newborns underwent routine NBS including orotic acid determination, 25 of whom were ultimately diagnosed with a UCD. Of 11 newborns with OTCD, orotate was elevated in seven but normal in two males with early-onset and two males with late-onset disease. Orotate was also elevated in archived dried blood spots of all seven retrospectively tested historical OTCD patients, only three of whom had originally been identified by NBS with low citrulline and elevated glutamine. Among the other UCDs emerge, three CPS1D cases and additional three retrospective CPS1D cases otherwise reported as a very rare condition. Combined levels of orotic acid and citrulline in routine NBS can enhance the detection of UCD, especially increasing the screening sensitivity for OTCD and differentiate it from CPS1D. Our data and the negligible extra cost for orotic acid determination might contribute to the discussion on screening for proximal UCDs in routine NBS.


Assuntos
Citrulina/sangue , Doença da Deficiência de Ornitina Carbomoiltransferase/diagnóstico , Ácido Orótico/sangue , Distúrbios Congênitos do Ciclo da Ureia/diagnóstico , Teste em Amostras de Sangue Seco , Feminino , Humanos , Recém-Nascido , Israel/epidemiologia , Masculino , Triagem Neonatal , Doença da Deficiência de Ornitina Carbomoiltransferase/epidemiologia , Estudos Retrospectivos , Distúrbios Congênitos do Ciclo da Ureia/epidemiologia
8.
Metab Brain Dis ; 36(4): 581-588, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33471299

RESUMO

Isolated defects in the mitochondrial respiratory chain complex II (CII; succinate-ubiquinone oxidoreductase) are extremely rare and mainly result from bi-allelic mutations in one of the nuclear encoded subunits: SDHA, SDHB and SDHD, which comprise CII and the assembly CII factor SDHAF1. We report an adolescent female who presented with global developmental delay, intellectual disability and childhood onset progressive bilateral optic atrophy. Whole exome sequencing of the patient and her unaffected parents identified the novel heterozygous de novo variant c.1984C > T [NM_004168.4] in the SDHA gene. Biochemical assessment of CII in the patient's derived fibroblasts and lymphocytes displayed considerably decreased CII residual activity compared with normal controls, when normalized to the integral mitochondrial enzyme citrate synthase. Protein modeling of the consequent p.Arg662Cys variant [NP-004159.2] suggested that this substitution will compromise the structural integrity of the FAD-binding protein at the C-terminus that will ultimately impair the FAD binding to SDHA, thus decreasing the entire CII activity. Our study emphasizes the role of certain heterozygous SDHA mutations in a distinct clinical phenotype dominated by optic atrophy and neurological impairment. This is the second mutation that has been reported to cause this phenotype. Furthermore, it adds developmental delay and cognitive disability to the expanding spectrum of the disorder. We propose to add SDHA to next generation sequencing gene panels of optic atrophy.


Assuntos
Disfunção Cognitiva/genética , Complexo II de Transporte de Elétrons/genética , Variação Genética/genética , Heterozigoto , Atrofia Óptica/genética , Adolescente , Sequência de Aminoácidos , Disfunção Cognitiva/complicações , Disfunção Cognitiva/diagnóstico por imagem , Complexo II de Transporte de Elétrons/química , Feminino , Humanos , Atrofia Óptica/complicações , Atrofia Óptica/diagnóstico por imagem , Estrutura Secundária de Proteína
9.
J Cell Sci ; 131(15)2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-30002135

RESUMO

AMPK-mTORC1 signaling senses nutrient availability, thereby regulating autophagy. Surprisingly, we found that, in ß-cells, the AMPK activator 5-amino-4-imidazolecarboxamide ribofuranoside (AICAR) inhibited, rather than stimulated, autophagy. AICAR is an intermediate in the generation of inosine monophosphate, with subsequent conversion to other purine nucleotides. Adenosine regulated autophagy in a concentration-dependent manner: at high concentrations, it mimicked the AICAR effect on autophagy, whereas at low concentrations it stimulated autophagy through its cognate A1 receptor. Adenosine regulation of autophagy was independent of AMPK or mTORC1 activity. Adenosine kinase (ADK) is the principal enzyme for metabolic adenosine clearance. ADK knockdown and pharmacological inhibition of the enzyme markedly stimulated autophagy in an adenosine A1 receptor-dependent manner. High-concentration adenosine increased insulin secretion in a manner sensitive to treatment with the autophagy inducer Tat-beclin1, and inhibition of autophagy augmented secretion. In conclusion, high concentrations of AICAR or adenosine inhibit autophagy, whereas physiological concentrations of adenosine or inhibition of adenosine clearance by ADK stimulate autophagy via the adenosine receptor. Adenosine might thus be an autocrine regulator of autophagy, independent of AMPK-mTORC1 signaling. Adenosine regulates insulin secretion, in part, through modulation of autophagy.


Assuntos
Nucleotídeos de Adenina/metabolismo , Autofagia/fisiologia , Células Secretoras de Insulina/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina , Animais , Western Blotting , Linhagem Celular , Imunofluorescência , Células Hep G2 , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais
10.
Neurobiol Dis ; 124: 57-66, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30423473

RESUMO

Mitochondrial malfunction is a common feature in advanced stages of neurodegenerative conditions, as is the case for the accumulation of aberrantly folded proteins, such as PrP in prion diseases. In this work, we investigated mitochondrial activity and expression of related factors vis a vis PrP accumulation at the subclinical stages of TgMHu2ME199K mice, modeling for genetic prion diseases. While these mice remain healthy until 5-6 months of age, they succumb to fatal disease at 12-14 months. We found that mitochondrial respiratory chain enzymatic activates and ATP/ROS production, were abnormally elevated in asymptomatic mice, concomitant with initial accumulation of disease related PrP. In parallel, the expression of Cytochrome c oxidase (COX) subunit IV isoform 1(Cox IV-1) was reduced and replaced by the activity of Cox IV isoform 2, which operates in oxidative neuronal conditions. At all stages of disease, Cox IV-1 was absent from cells accumulating disease related PrP, suggesting that PrP aggregates may directly compromise normal mitochondrial function. Administration of Nano-PSO, a brain targeted antioxidant, to TgMHu2ME199K mice, reversed functional and biochemical mitochondrial functions to normal conditions regardless of the presence of misfolded PrP. Our results therefore indicate that in genetic prion disease, oxidative damage initiates long before clinical manifestations. These manifest only when aggregated PrP levels are too high for the compensatory mechanisms to sustain mitochondrial activity.


Assuntos
Mitocôndrias/enzimologia , Doenças Priônicas/enzimologia , Doenças Priônicas/genética , Proteínas Priônicas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/administração & dosagem , Modelos Animais de Doenças , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Óleos de Plantas/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo
11.
Am J Hum Genet ; 99(4): 860-876, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27693233

RESUMO

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.


Assuntos
Translocador 1 do Nucleotídeo Adenina/genética , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/genética , Genes Dominantes/genética , Doenças Mitocondriais/genética , Mutação , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Idade de Início , Arilamina N-Acetiltransferase/genética , Criança , Pré-Escolar , Transporte de Elétrons/genética , Exoma/genética , Feminino , Humanos , Lactente , Recém-Nascido , Isoenzimas/genética , Masculino , Doenças Mitocondriais/patologia , Músculo Esquelético/metabolismo
12.
J Cell Sci ; 130(8): 1486-1493, 2017 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-28280117

RESUMO

Sphingolipids modulate clathrin-mediated endocytosis (CME) by altering the biophysical properties of membranes. We now examine CME in astrocytes cultured from ceramide synthase 2 (CerS2) null mice, which have an altered sphingolipid acyl chain composition. The rate of endocytosis of low-density lipoprotein and transferrin, which are internalized via CME, was reduced in CerS2 null astrocytes, although the rate of caveolin-mediated endocytosis was unaltered. Levels of clathrin heavy chain were increased, which was due to decreased levels of Hsc70 (also known as HSPA8), a protein involved in clathrin uncoating. Hsc70 levels were decreased because of lower levels of binding of Sp1 to position -68 in the Hsc70 promoter. Levels of Sp1 were downregulated due to oxidative stress, which was elevated fourfold in CerS2 null astrocytes. Furthermore, induction of oxidative stress in wild-type astrocytes decreased the rate of CME, whereas amelioration of oxidative stress in CerS2 null astrocytes reversed the decrease. Our data are consistent with the notion that sphingolipids not only change membrane biophysical properties but also that changes in their composition can result in downstream effects that indirectly impinge upon a number of cellular pathways, such as CME.


Assuntos
Astrócitos/fisiologia , Ceramidas/metabolismo , Endocitose , Fígado/fisiologia , Estresse Oxidativo/imunologia , Esfingolipídeos/metabolismo , Esfingosina N-Aciltransferase/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Ceramidas/química , Clatrina/metabolismo , Endocitose/genética , Camundongos , Camundongos Knockout , Estresse Oxidativo/genética , Engenharia de Proteínas , Transdução de Sinais , Esfingosina N-Aciltransferase/genética
13.
J Hum Genet ; 64(6): 589-595, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30903008

RESUMO

Translation of mitochondrial-specific DNA is required for proper mitochondrial function and energy production. For this purpose, an elaborate network of dedicated molecular machinery including initiation, elongation and termination factors exists. We describe a patient with an unusual phenotype and a novel homozygous missense variant in TUFM (c.344A>C; p.His115Pro), encoding mtDNA translation elongating factor Tu (EFTu). To date, only four patients have been reported with bi-allelic mutations in TUFM, leading to combined oxidative phosphorylation deficiency 4 (COXPD4) characterized by severe early-onset lactic acidosis and progressive fatal infantile encephalopathy. The patient presented here expands the phenotypic features of TUFM-related disease, exhibiting lactic acidosis and dilated cardiomyopathy without progressive encephalopathy. This warrants the inclusion of TUFM in differential diagnosis of metabolic cardiomyopathy. Cases that further refine genotype-phenotype associations and characterize the molecular basis of mitochondrial disorders allow clinicians to predict disease prognosis, greatly impacting patient care, as well as provide families with reproductive planning options.


Assuntos
Acidose Láctica/genética , Cardiomiopatias/genética , Erros Inatos do Metabolismo/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Fator Tu de Elongação de Peptídeos/genética , Acidose Láctica/fisiopatologia , Sequência de Aminoácidos/genética , Cardiomiopatias/fisiopatologia , Consanguinidade , DNA Mitocondrial/genética , Feminino , Homozigoto , Humanos , Lactente , Masculino , Erros Inatos do Metabolismo/fisiopatologia , Doenças Mitocondriais/fisiopatologia , Mutação , Fosforilação Oxidativa , Sequenciamento do Exoma
14.
Neurochem Res ; 44(10): 2372-2384, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30968303

RESUMO

Primary deficiency of coenzyme Q10 (CoQ10 ubiquinone), is classified as a mitochondrial respiratory chain disorder with phenotypic variability. The clinical manifestation may involve one or multiple tissue with variable severity and presentation may range from infancy to late onset. ADCK3 gene mutations are responsible for the most frequent form of hereditary CoQ10 deficiency (Q10 deficiency-4 OMIM #612016) which is mainly associated with autosomal recessive spinocerebellar ataxia (ARCA2, SCAR9). Here we provide the clinical, biochemical and genetic investigation for unrelated three nuclear families presenting an autosomal form of Spino-Cerebellar Ataxia due to novel mutations in the ADCK3 gene. Using next generation sequence technology we identified a homozygous Gln343Ter mutation in one family with severe, early onset of the disease and compound heterozygous mutations of Gln343Ter and Ser608Phe in two other families with variable manifestations. Biochemical investigation in fibroblasts showed decreased activity of the CoQ dependent mitochondrial respiratory chain enzyme succinate cytochrome c reductase (complex II + III). Exogenous CoQ slightly improved enzymatic activity, ATP production and decreased oxygen free radicals in some of the patient's cells. Our results are presented in comparison to previously reported mutations and expanding the clinical, molecular and biochemical spectrum of ADCK3 related CoQ10 deficiencies.


Assuntos
Ataxia/genética , Fibroblastos/metabolismo , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Debilidade Muscular/genética , Ubiquinona/análogos & derivados , Ubiquinona/deficiência , Ataxia Cerebelar/genética , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Mutação/genética , Ubiquinona/genética
15.
Metab Brain Dis ; 34(2): 557-563, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30637540

RESUMO

D-glycerate 2 kinase (DGK) is an enzyme that mediates the conversion of D-glycerate, an intermediate metabolite of serine and fructose metabolism, to 2-phosphoglycerate. Deficiency of DGK leads to accumulation of D-glycerate in various tissues and its massive excretion in urine. D-glyceric aciduria (DGA) is an autosomal recessive metabolic disorder caused by mutations in the GLYCTK gene. The clinical spectrum of DGA is highly variable, ranging from severe progressive infantile encephalopathy to a practically asymptomatic condition. We describe a male patient from a consanguineous Arab family with infantile onset of DGA, characterized by profound psychomotor retardation, progressive microcephaly, intractable seizures, cortical blindness and deafness. Consecutive brain MR imaging showed an evolving brain atrophy, thinning of the corpus callosum and diffuse abnormal white matter signals. Whole exome sequencing identified the homozygous missense variant in the GLYCTK gene [c.455 T > C, NM_145262.3], which affected a highly conserved leucine residue located at a domain of yet unknown function of the enzyme [p.Leu152Pro, NP_660305]. In silico analysis of the variant supported its pathogenicity. A review of the 15 previously reported patients, together with the current one, confirms a clear association between DGA and severe neurological impairment. Yet, future studies of additional patients with DGA are required to better understand the clinical phenotype and pathogenesis.


Assuntos
Encefalopatias/metabolismo , Epilepsia/metabolismo , Hiperoxalúria Primária/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Encefalopatias/genética , Criança , Epilepsia/diagnóstico , Epilepsia/genética , Ácidos Glicéricos/metabolismo , Humanos , Hiperoxalúria Primária/genética , Lactente , Masculino , Mutação/genética , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Espasmos Infantis/genética , Espasmos Infantis/metabolismo
16.
Biochim Biophys Acta Bioenerg ; 1859(9): 893-900, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29886046

RESUMO

Mitochondrial cytochrome c oxidase (COX, respiratory chain complex IV), contributes to ATP production via oxidative phosphorylation (OXPHOS). Clinical presentation of COX deficiency is heterogeneous ranging from mild to severe neuromuscular diseases. Anemia is among the symptoms and we have previously reported Fanconi anemia like features in COX4-1 deficiency, suggesting genomic instability and our preliminary results detected nuclear double stranded DNA breaks (DSB). We now quantified the DSB by phospho histone H2AX Ser139 staining of COX4-1 and COX6B1 deficient fibroblasts (225% and 215% of normal, respectively) and confirmed their occurrence by neutral comet assay. We further explored the mechanism of DNA damage by studying normal fibroblasts treated with micromolar concentrations of cyanide (KCN). Present results demonstrate elevated nuclear DSB in cells treated with 50 µM KCN for 24 h (170% of normal) in high-glucose medium conditions where ROS and ATP remain normal, although Glutathione content was partially decreased. In glucose-free and serum-free medium, where growth is hampered, DSB were not elevated. Additionally we demonstrate the benefit of nicotinamide riboside (NR) which ameliorated DSB in COX4-1, COX6B1 and KCN treated cells (130%, 154% and 87% of normal cells, respectively). Conversely a negative effect of a poly[ADP-ribose] polymerase (PARP) inhibitor was found. Although additional investigation is needed, our findings raise the possibility that the pathomechanism of COX deficiency and possibly also in other OXPHOS defects, include nuclear DNA damage resulting from nicotinamide adenine dinucleotide (NAD+) deficit combined with a replicative state, rather than oxidative stress and energy depletion.


Assuntos
Núcleo Celular/genética , Deficiência de Citocromo-c Oxidase/genética , Deficiência de Citocromo-c Oxidase/patologia , Dano ao DNA , Complexo IV da Cadeia de Transporte de Elétrons/genética , Fibroblastos/patologia , Mitocôndrias/patologia , Células Cultivadas , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Fibroblastos/metabolismo , Humanos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Estresse Oxidativo
17.
Genet Med ; 20(4): 444-451, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29261183

RESUMO

PurposeDiagnosing primary mitochondrial diseases (MDs) is challenging in clinical practice. The mitochondrial disease criteria (MDC) have been developed to quantify the clinical picture and evaluate the probability of an underlying MD and the need for a muscle biopsy. In this new genetic era with next-generation sequencing in routine practice, we aim to validate the diagnostic value of MDC.MethodsWe retrospectively studied MDC in a multicenter cohort of genetically confirmed primary MD patients.ResultsWe studied 136 patients (61 male, 91 nuclear DNA (nDNA) mutations). Forty-five patients (33%) had probable MD and 69 (51%) had definite MD according to the MDC. A muscle biopsy was performed in 63 patients (47%). Patients with nDNA mutations versus mitochondrial DNA mutations were younger (6.4 ± 9.7 versus 19.5 ± 17.3 y) and had higher MDC (7.07 ± 1.12/8 versus 5.69 ± 1.94/8). At a cutoff of 6.5/8, the sensitivity to diagnose patients with nDNA mutations is 72.5% with a positive predictive value of 69.5%. In the nDNA mutation group, whole-exome sequencing could diagnose patients with lower scores (MDC (6.84 ± 1.51/8) compared to Sanger sequencing MDC (7.44 ± 1.13/8, P = 0.025)). Moreover 7/8 patients diagnosed with possible MD by MDC were diagnosed by whole-exome sequencing.ConclusionMDC remain very useful in the clinical diagnosis of MD, in interpreting whole-exome results and deciding on the need for performing muscle biopsy.


Assuntos
Genoma Mitocondrial , Genômica , Mitocôndrias/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Genes Mitocondriais , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Genômica/métodos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mutação , Estudos Retrospectivos , Fluxo de Trabalho , Adulto Jovem
18.
Genet Med ; 20(9): 1004-1012, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29300381

RESUMO

PURPOSE: We aimed to identify the cause of disease in patients suffering from a distinctive, atypical form of Usher syndrome. METHODS: Whole-exome and genome sequencing were performed in five patients from three families of Yemenite Jewish origin, suffering from distinctive retinal degeneration phenotype and sensorineural hearing loss. Functional analysis of the wild-type and mutant proteins was performed in human fibrosarcoma cells. RESULTS: We identified a homozygous founder missense variant, c.133G>T (p.D45Y) in arylsulfatase G (ARSG). All patients shared a distinctive retinal phenotype with ring-shaped atrophy along the arcades engirdling the fovea, resulting in ring scotoma. In addition, patients developed moderate to severe sensorineural hearing loss. Both vision and hearing loss appeared around the age of 40 years. The identified variant affected a fully conserved amino acid that is part of the catalytic site of the enzyme. Functional analysis of the wild-type and mutant proteins showed no basal activity of p.D45Y. CONCLUSION: Homozygosity for ARSG-p.D45Y in humans leads to protein dysfunction, causing an atypical combination of late-onset Usher syndrome. Although there is no evidence for generalized clinical manifestations of lysosomal storage diseases in this set of patients, we cannot rule out the possibility that mild and late-onset symptoms may appear.


Assuntos
Arilsulfatases/genética , Síndromes de Usher/genética , Adulto , Arilsulfatases/metabolismo , Sequência de Bases , Análise Mutacional de DNA , Feminino , Efeito Fundador , Homozigoto , Humanos , Masculino , Mutação , Mutação de Sentido Incorreto , Linhagem , Retina/metabolismo , Degeneração Retiniana/enzimologia , Degeneração Retiniana/genética , Retinose Pigmentar/enzimologia , Retinose Pigmentar/genética , Sequenciamento do Exoma , Sequenciamento Completo do Genoma
19.
Am J Med Genet A ; 176(2): 330-336, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29226520

RESUMO

SLC25A1 mutations are associated with combined D,L-2-hydroxyglutaric aciduria (DL- 2HGA; OMIM #615182), characterized by muscular hypotonia, severe neurodevelopmental dysfunction and intractable seizures. SLC25A1 encodes the mitochondrial citrate carrier (CIC), which mediates efflux of the mitochondrial tricarboxylic acid (TCA) cycle intermediates citrate and isocitrate in exchange for cytosolic malate. Only a single family with an SLC25A1 mutation has been described in which mitochondrial respiratory chain dysfunction was documented, specifically in complex IV. Five infants of two consanguineous Bedouin families of the same tribe presented with small head circumference and neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development culminating in early death. Ventricular septal defects (VSD) were demonstrated in three patients. Blood and CSF lactate were elevated with normal levels of plasma amino acids and free carnitine and increased 2-OH-glutaric acid urinary exertion. EEG was compatible with white matter disorder. Brain MRI revealed ventriculomegaly, thin corpus callosum with increased lactate peak on spectroscopy. Mitochondrial complex V deficiency was demonstrated in skeletal muscle biopsy of one infant. Homozygosity mapping and sequencing ruled out homozygosity of affected individuals in all known complex V-associated genes. Whole exome sequencing identified a novel homozygous SLC25A1 c.713A>G (p.Asn238Ser) mutation, segregating as expected in the affected kindred and not found in 220 control alleles. Thus, SLC25A1 mutations might be associated with mitochondrial complex V deficiency and should be considered in the differential diagnosis of mitochondrial respiratory chain defects.


Assuntos
Proteínas de Transporte de Ânions/genética , Homozigoto , Mitocôndrias/genética , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação , Adolescente , Adulto , Encéfalo/anormalidades , Encéfalo/diagnóstico por imagem , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Transportadores de Ânions Orgânicos , Linhagem , Fenótipo , Adulto Jovem
20.
PLoS Genet ; 11(8): e1005388, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26247364

RESUMO

Calmodulin lysine methyl transferase (CaM KMT) is ubiquitously expressed and highly conserved from plants to vertebrates. CaM is frequently trimethylated at Lys-115, however, the role of CaM methylation in vertebrates has not been studied. CaM KMT was found to be homozygously deleted in the 2P21 deletion syndrome that includes 4 genes. These patients present with cystinuria, severe intellectual disabilities, hypotonia, mitochondrial disease and facial dysmorphism. Two siblings with deletion of three of the genes included in the 2P21 deletion syndrome presented with cystinuria, hypotonia, a mild/moderate mental retardation and a respiratory chain complex IV deficiency. To be able to attribute the functional significance of the methylation of CaM in the mouse and the contribution of CaM KMT to the clinical presentation of the 2p21deletion patients, we produced a mouse model lacking only CaM KMT with deletion borders as in the human 2p21deletion syndrome. No compensatory activity for CaM methylation was found. Impairment of complexes I and IV, and less significantly III, of the mitochondrial respiratory chain was more pronounced in the brain than in muscle. CaM KMT is essential for normal body growth and somatosensory development, as well as for the proper functioning of the adult mouse brain. Developmental delay was demonstrated for somatosensory function and for complex behavior, which involved both basal motor function and motivation. The mutant mice also had deficits in motor learning, complex coordination and learning of aversive stimuli. The mouse model contributes to the evaluation of the role of methylated CaM. CaM methylation appears to have a role in growth, muscle strength, somatosensory development and brain function. The current study has clinical implications for human patients. Patients presenting slow growth and muscle weakness that could result from a mitochondrial impairment and mental retardation should be considered for sequence analysis of the CaM KMT gene.


Assuntos
Metiltransferases/fisiologia , Animais , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Deleção Cromossômica , Retroalimentação Sensorial , Feminino , Masculino , Metilação , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Força Muscular , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Processamento de Proteína Pós-Traducional
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