RESUMO
Argininosuccinate lyase deficiency (ASLD) is a recessive metabolic disorder caused by variants in ASL. In an essential step in urea synthesis, ASL breaks down argininosuccinate (ASA), a pathognomonic ASLD biomarker. The severe disease forms lead to hyperammonemia, neurological injury, and even early death. The current treatments are unsatisfactory, involving a strict low-protein diet, arginine supplementation, nitrogen scavenging, and in some cases, liver transplantation. An unmet need exists for improved, efficient therapies. Here, we show the potential of a lipid nanoparticle-mediated CRISPR approach using adenine base editors (ABEs) for ASLD treatment. To model ASLD, we first generated human-induced pluripotent stem cells (hiPSCs) from biopsies of individuals homozygous for the Finnish founder variant (c.1153C>T [p.Arg385Cys]) and edited this variant using the ABE. We then differentiated the hiPSCs into hepatocyte-like cells that showed a 1,000-fold decrease in ASA levels compared to those of isogenic non-edited cells. Lastly, we tested three different FDA-approved lipid nanoparticle formulations to deliver the ABE-encoding RNA and the sgRNA targeting the ASL variant. This approach efficiently edited the ASL variant in fibroblasts with no apparent cell toxicity and minimal off-target effects. Further, the treatment resulted in a significant decrease in ASA, to levels of healthy donors, indicating restoration of the urea cycle. Our work describes a highly efficient approach to editing the disease-causing ASL variant and restoring the function of the urea cycle. This method relies on RNA delivered by lipid nanoparticles, which is compatible with clinical applications, improves its safety profile, and allows for scalable production.
Assuntos
Argininossuccinato Liase , Acidúria Argininossuccínica , Humanos , Argininossuccinato Liase/genética , Acidúria Argininossuccínica/genética , Acidúria Argininossuccínica/terapia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA Guia de Sistemas CRISPR-Cas , Ureia , Edição de Genes/métodosRESUMO
Mitochondrial acyl-coenzyme A species are emerging as important sources of protein modification and damage. Succinyl-CoA ligase (SCL) deficiency causes a mitochondrial encephalomyopathy of unknown pathomechanism. Here, we show that succinyl-CoA accumulates in cells derived from patients with recessive mutations in the tricarboxylic acid cycle (TCA) gene succinyl-CoA ligase subunit-ß (SUCLA2), causing global protein hyper-succinylation. Using mass spectrometry, we quantify nearly 1,000 protein succinylation sites on 366 proteins from patient-derived fibroblasts and myotubes. Interestingly, hyper-succinylated proteins are distributed across cellular compartments, and many are known targets of the (NAD+)-dependent desuccinylase SIRT5. To test the contribution of hyper-succinylation to disease progression, we develop a zebrafish model of the SCL deficiency and find that SIRT5 gain-of-function reduces global protein succinylation and improves survival. Thus, increased succinyl-CoA levels contribute to the pathology of SCL deficiency through post-translational modifications.
Assuntos
Acil Coenzima A/metabolismo , Doenças Mitocondriais/patologia , Succinato-CoA Ligases/genética , Animais , Células Cultivadas , Feminino , Humanos , Lactente , Lisina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Mutação , Proteômica , Sirtuínas/deficiência , Sirtuínas/genética , Sirtuínas/metabolismo , Succinato-CoA Ligases/deficiência , Succinato-CoA Ligases/metabolismo , Análise de Sobrevida , Peixe-ZebraRESUMO
Mitochondrial dysfunction elicits stress responses that safeguard cellular homeostasis against metabolic insults. Mitochondrial integrated stress response (ISRmt) is a major response to mitochondrial (mt)DNA expression stress (mtDNA maintenance, translation defects), but the knowledge of dynamics or interdependence of components is lacking. We report that in mitochondrial myopathy, ISRmt progresses in temporal stages and development from early to chronic and is regulated by autocrine and endocrine effects of FGF21, a metabolic hormone with pleiotropic effects. Initial disease signs induce transcriptional ISRmt (ATF5, mitochondrial one-carbon cycle, FGF21, and GDF15). The local progression to 2nd metabolic ISRmt stage (ATF3, ATF4, glucose uptake, serine biosynthesis, and transsulfuration) is FGF21 dependent. Mitochondrial unfolded protein response marks the 3rd ISRmt stage of failing tissue. Systemically, FGF21 drives weight loss and glucose preference, and modifies metabolism and respiratory chain deficiency in a specific hippocampal brain region. Our evidence indicates that FGF21 is a local and systemic messenger of mtDNA stress in mice and humans with mitochondrial disease.
Assuntos
DNA Mitocondrial/metabolismo , Fatores de Crescimento de Fibroblastos/fisiologia , Mitocôndrias/metabolismo , Miopatias Mitocondriais/metabolismo , Estresse Fisiológico/fisiologia , Fatores Ativadores da Transcrição/metabolismo , Animais , Linhagem Celular , DNA Mitocondrial/genética , Escherichia coli , Feminino , Fatores de Crescimento de Fibroblastos/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Masculino , Camundongos , Mitocôndrias/genética , Miopatias Mitocondriais/genética , Deleção de Sequência , Estresse Fisiológico/genéticaRESUMO
Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.
Assuntos
Alanina-tRNA Ligase/metabolismo , Cardiomiopatias/enzimologia , Alanina-tRNA Ligase/genética , Cardiomiopatias/genética , Doenças em Gêmeos/genética , Estabilidade Enzimática , Fibroblastos/metabolismo , Genes Recessivos , Humanos , Lactente , Ácido Láctico , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Linhagem , Insuficiência Respiratória/enzimologiaRESUMO
Mitochondria are central organelles to cellular metabolism. Their function relies largely on nuclear-encoded proteins that must be imported from the cytosol, and thus the protein import pathways are important for the maintenance of mitochondrial proteostasis. Mitochondrial HSP70 (mtHsp70) is a key component in facilitating the translocation of proteins through the inner membrane into the mitochondrial matrix. Its protein folding cycle is regulated by the nucleotide-exchange factor GrpE, which triggers the release of folded proteins by ATP rebinding. Vertebrates have two mitochondrial GrpE paralogs, GRPEL1 and 2, but without clearly defined roles. Using BioID proximity labeling to identify potential binding partners of the GRPELs in the mitochondrial matrix, we obtained results supporting a model where both GRPELs regulate mtHsp70 as homodimers. We show that GRPEL2 is not essential in human cultured cells, and its absence does not prevent mitochondrial protein import. Instead we find that GRPEL2 is redox regulated in oxidative stress. In the presence of hydrogen peroxide, GRPEL2 forms dimers through intermolecular disulfide bonds in which Cys87 is the thiol switch. We propose that the dimerization of GRPEL2 may activate the folding machinery responsible for protein import into mitochondrial matrix or enhance the chaperone activity of mtHSP70, thus protecting mitochondrial proteostasis in oxidative stress.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Linhagem Celular , Proteínas de Choque Térmico HSP70/análise , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/metabolismo , Oxirredução , Dobramento de Proteína , Multimerização Proteica , Transporte ProteicoRESUMO
The folate cycle is an essential metabolic pathway in the cell, involved in nucleotide synthesis, maintenance of the redox balance in the cell, methionine metabolism and re-methylation reactions. Standardised methods for the measurement of folate cycle intermediates in different biological matrices are in great demand. Here we describe a rapid, sensitive, precise and accurate liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method with a wide calibration curve range and a short run time for the simultaneous determination of folate cycle metabolites, including tetrahydrofolic acid (THF), 5methyl THF, 5formyl THF, 5,10methenyl THF, 5,10methylene THF, dihydrofolic acid (DHF) and folic acid in different biological matrices. Extraction of folate derivatives from soft and hard tissue samples as well as from adherent cells was achieved using homogenisation in buffer, while extraction from the whole blood and plasma relied on the anion exchange solid-phase extraction (SPE) method. Chromatographic separation was completed using a Waters Atlantis dC18 2.0â¯×â¯100â¯mm, 3-µ column with a gradient elution using formic acid in water (0.1% v/v) and acetonitrile as the mobile phases. LC gradient started with 95% of the aqueous phase which was gradually changed to 95% of the organic phase during 2.70â¯min in order to separate the selected metabolites. The analytes were separated with a run time of 5â¯min at a flow rate of 0.300â¯mL/min and detected using a Waters Xevo-TQS triple quadrupole mass spectrometer in the multiple reaction monitoring mode (MRM) at positive polarity. The instrument response was linear over a calibration range of 0.5 to 2500â¯ng/mL (r2â¯>â¯0.980). The developed bioanalytical method was thoroughly validated in terms of accuracy, precision, linearity, recovery, sensitivity and stability for tissue and blood samples. The matrix effect was compensated by using structurally similar isotope labelled internal standard (IS), 13C5methyl THF, for all folate metabolites. However, not all folate metabolites can be accurately quantified using this method due to their high interconversion rates especially at low pH. This applies to 5,10methylene THF which interconverts into THF, and 5,10methenylTHF interconverting into 5formylTHF. Using this method, we measured folate cycle intermediates in mouse bone marrow cells and plasma, in human whole blood; in mouse muscle, liver, heart and brain samples.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácido Fólico/análise , Espectrometria de Massas em Tandem/métodos , Animais , Química Encefálica , Ácido Fólico/sangue , Ácido Fólico/química , Ácido Fólico/isolamento & purificação , Modelos Lineares , Músculos/química , Miocárdio/química , Especificidade de Órgãos , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Mitochondrial dysfunction manifests as different neurological diseases, but the mechanisms underlying the clinical variability remain poorly understood. To clarify whether different brain cells have differential sensitivity to mitochondrial dysfunction, we induced mitochondrial DNA (mtDNA) depletion in either neurons or astrocytes of mice, by inactivating Twinkle (TwKO), the replicative mtDNA helicase. Here we show that astrocytes, the most abundant cerebral cell type, are chronically activated upon mtDNA loss, leading to early-onset spongiotic degeneration of brain parenchyma, microgliosis and secondary neurodegeneration. Neuronal mtDNA loss does not, however, cause symptoms until 8 months of age. Findings in astrocyte-TwKO mimic neuropathology of Alpers syndrome, infantile-onset mitochondrial spongiotic encephalopathy caused by mtDNA maintenance defects. Our evidence indicates that (1) astrocytes are dependent on mtDNA integrity; (2) mitochondrial metabolism contributes to their activation; (3) chronic astrocyte activation has devastating consequences, underlying spongiotic encephalopathy; and that (4) astrocytes are a potential target for interventions.
Assuntos
Astrócitos/metabolismo , Encefalopatias/genética , DNA Mitocondrial/genética , Doenças Mitocondriais/genética , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Mitocondrial/metabolismo , Camundongos Knockout , Microscopia Eletrônica , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutação , Neurônios/metabolismoRESUMO
Accuracy of protein synthesis is enabled by the selection of amino acids for tRNA charging by aminoacyl-tRNA synthetases (ARSs), and further enhanced by the proofreading functions of some of these enzymes for eliminating tRNAs mischarged with noncognate amino acids. Mouse models of editing-defective cytoplasmic alanyl-tRNA synthetase (AlaRS) have previously demonstrated the importance of proofreading for cytoplasmic protein synthesis, with embryonic lethal and progressive neurodegeneration phenotypes. Mammalian mitochondria import their own set of nuclear-encoded ARSs for translating critical polypeptides of the oxidative phosphorylation system, but the importance of editing by the mitochondrial ARSs for mitochondrial proteostasis has not been known. We demonstrate here that the human mitochondrial AlaRS is capable of editing mischarged tRNAs in vitro, and that loss of the proofreading activity causes embryonic lethality in mice. These results indicate that tRNA proofreading is essential in mammalian mitochondria, and cannot be overcome by other quality control mechanisms.
Assuntos
Alanina-tRNA Ligase/genética , Mitocôndrias/genética , Edição de RNA , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/genética , Alanina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Mamíferos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mutação , Biossíntese de Proteínas/genética , RNA de Transferência/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Leigh syndrome is a severe infantile encephalopathy with an exceptionally variable genetic background. We studied the exome of a child manifesting with Leigh syndrome at one month of age and progressing to death by the age of 2.4 years, and identified novel compound heterozygous variants in PNPT1, encoding the polynucleotide phosphorylase (PNPase). Expression of the wild type PNPT1 in the subject's myoblasts functionally complemented the defects, and the pathogenicity was further supported by structural predictions and protein and RNA analyses. PNPase is a key enzyme in mitochondrial RNA metabolism, with suggested roles in mitochondrial RNA import and degradation. The variants were predicted to locate in the PNPase active site and disturb the RNA processing activity of the enzyme. The PNPase trimer formation was not affected, but specific RNA processing intermediates derived from mitochondrial transcripts of the ND6 subunit of Complex I, as well as small mRNA fragments, accumulated in the subject's myoblasts. Mitochondrial RNA processing mediated by the degradosome consisting of hSUV3 and PNPase is poorly characterized, and controversy on the role and location of PNPase within human mitochondria exists. Our evidence indicates that PNPase activity is essential for the correct maturation of the ND6 transcripts, and likely for the efficient removal of degradation intermediates. Loss of its activity will result in combined respiratory chain deficiency, and a classic respiratory chain-deficiency-associated disease, Leigh syndrome, indicating an essential role for the enzyme for normal function of the mitochondrial respiratory chain.
Assuntos
Exorribonucleases/genética , Exorribonucleases/metabolismo , Doença de Leigh/genética , Pré-Escolar , Exoma , Exorribonucleases/química , Feminino , Expressão Gênica , Humanos , Lactente , Recém-Nascido , Doença de Leigh/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Polirribonucleotídeo Nucleotidiltransferase , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA MitocondrialRESUMO
DNA polymerase γ (Pol γ) is a key component of the mitochondrial DNA replisome and an important cause of neurological diseases. Despite the availability of its crystal structures, the molecular mechanism of DNA replication, the switch between polymerase and exonuclease activities, the site of replisomal interactions, and functional effects of patient mutations that do not affect direct catalysis have remained elusive. Here we report the first atomistic classical molecular dynamics simulations of the human Pol γ replicative complex. Our simulation data show that DNA binding triggers remarkable changes in the enzyme structure, including (1) completion of the DNA-binding channel via a dynamic subdomain, which in the apo form blocks the catalytic site, (2) stabilization of the structure through the distal accessory ß-subunit, and (3) formation of a putative transient replisome-binding platform in the "intrinsic processivity" subdomain of the enzyme. Our data indicate that noncatalytic mutations may disrupt replisomal interactions, thereby causing Pol γ-associated neurodegenerative disorders.
Assuntos
DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Mitocôndrias/enzimologia , Simulação de Dinâmica Molecular , Domínio Catalítico , DNA/metabolismo , DNA Polimerase gama , Humanos , Mutação , Doenças Neurodegenerativas/enzimologia , Doenças Neurodegenerativas/genética , Estrutura Secundária de Proteína , RotaçãoRESUMO
De novo mutations in ATAD3A (ATPase family AAA-domain containing protein 3A) were recently found to cause a neurological syndrome with developmental delay, hypotonia, spasticity, optic atrophy, axonal neuropathy, and hypertrophic cardiomyopathy. Using whole-exome sequencing, we identified a dominantly inherited heterozygous variant c.1064G > A (p.G355D) in ATAD3A in a mother presenting with hereditary spastic paraplegia (HSP) and axonal neuropathy and her son with dyskinetic cerebral palsy, both with disease onset in childhood. HSP is a clinically and genetically heterogeneous disorder of the upper motor neurons. Symptoms beginning in early childhood may resemble spastic cerebral palsy. The function of ATAD3A, a mitochondrial inner membrane AAA ATPase, is yet undefined. AAA ATPases form hexameric rings, which are catalytically dependent on the co-operation of the subunits. The dominant-negative patient mutation affects the Walker A motif, which is responsible for ATP binding in the AAA module of ATAD3A, and we show that the recombinant mutant ATAD3A protein has a markedly reduced ATPase activity. We further show that overexpression of the mutant ATAD3A fragments the mitochondrial network and induces lysosome mass. Similarly, we observed altered dynamics of the mitochondrial network and increased lysosomes in patient fibroblasts and neurons derived through differentiation of patient-specific induced pluripotent stem cells. These alterations were verified in patient fibroblasts to associate with upregulated basal autophagy through mTOR inactivation, resembling starvation. Mutations in ATAD3A can thus be dominantly inherited and underlie variable neurological phenotypes, including HSP, with intrafamiliar variability. This finding extends the group of mitochondrial inner membrane AAA proteins associated with spasticity.
Assuntos
Adenosina Trifosfatases/genética , Paralisia Cerebral/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Paraplegia Espástica Hereditária/genética , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/biossíntese , Adolescente , Adulto , Axônios/metabolismo , Axônios/patologia , Paralisia Cerebral/patologia , Pré-Escolar , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Proteínas de Membrana/biossíntese , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Dinâmica Mitocondrial/genética , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Proteínas Mitocondriais/biossíntese , Mutação , Paraplegia Espástica Hereditária/patologia , Serina-Treonina Quinases TOR/genéticaRESUMO
Isolated defects of the mitochondrial respiratory complex II (succinate dehydrogenase, SDH) are rare, accounting for approximately 2% of all respiratory chain deficiency diagnoses. Here, we report clinical and molecular investigations of three family members with a heterozygous mutation in the large flavoprotein subunit SDHA previously described to cause complex II deficiency. The index patient presented with bilateral optic atrophy and ocular movement disorder, a progressive polyneuropathy, psychiatric involvement, and cardiomyopathy. Two of his children presented with cardiomyopathy and methylglutaconic aciduria in early childhood. The daughter deceased at the age of 7 months due to cardiac insufficiency. The 30-year old son presents with cardiomyopathy and developed bilateral optic atrophy in adulthood. Of the four nuclear encoded proteins composing complex II (SDHA, SDHB, SDHC, SDHD) and currently known assembly factors SDHAF1 and SDHAF2 mainly recessively inherited mutations have been described in SDHA, SDHB, SDHD, and SDHAF1 to be causative for mitochondrial disease phenotypes. This is the second report presenting autosomal dominant inheritance of a SDHA mutation.© 2016 Wiley Periodicals, Inc.
Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Complexo II de Transporte de Elétrons/deficiência , Mutação , Fenótipo , Succinato Desidrogenase/genética , Adolescente , Alelos , Substituição de Aminoácidos , Biomarcadores , Códon , Análise Mutacional de DNA , Evolução Fatal , Feminino , Genes Mitocondriais , Genótipo , Humanos , Masculino , Modelos Moleculares , Linhagem , Conformação Proteica , Succinato Desidrogenase/químicaRESUMO
Mitochondrial dysfunction affects cellular energy metabolism, but less is known about the consequences for cytoplasmic biosynthetic reactions. We report that mtDNA replication disorders caused by TWINKLE mutations-mitochondrial myopathy (MM) and infantile onset spinocerebellar ataxia (IOSCA)-remodel cellular dNTP pools in mice. MM muscle shows tissue-specific induction of the mitochondrial folate cycle, purine metabolism, and imbalanced and increased dNTP pools, consistent with progressive mtDNA mutagenesis. IOSCA-TWINKLE is predicted to hydrolyze dNTPs, consistent with low dNTP pools and mtDNA depletion in the disease. MM muscle also modifies the cytoplasmic one-carbon cycle, transsulfuration, and methylation, as well as increases glucose uptake and its utilization for de novo serine and glutathione biosynthesis. Our evidence indicates that the mitochondrial replication machinery communicates with cytoplasmic dNTP pools and that upregulation of glutathione synthesis through glucose-driven de novo serine biosynthesis contributes to the metabolic stress response. These results are important for disorders with primary or secondary mtDNA instability and offer targets for metabolic therapy.
Assuntos
DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Miopatias Mitocondriais/metabolismo , Nucleotídeos/metabolismo , Degenerações Espinocerebelares/metabolismo , Adulto , Animais , Carbono/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Mitocondrial/genética , Feminino , Ácido Fólico/metabolismo , Glucose/metabolismo , Glutationa/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/genética , Mitocôndrias/patologia , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Mutação , Serina/metabolismo , Degenerações Espinocerebelares/genética , Degenerações Espinocerebelares/patologiaRESUMO
We report a 10-year-old girl presenting with severe neonatal hypertrophic cardiomyopathy (HCM), feeding difficulties, mildly abnormal facial features, and progressive skeletal muscle symptoms but with normal cognitive development. Targeted oligonucleotide-selective sequencing of 101 cardiomyopathy genes revealed the genetic diagnosis, and the mutation was verified by Sanger sequencing in the patient and her parents. To offer insights into the potential mechanism of patient mutation, protein structural analysis was performed using the resolved structure of human activated HRAS protein with bound GTP analogue (PDB id 5P21) in Discovery Studio 4.5 (Dassault Systèmes Biovia, San Diego, CA). The patient with hypertrophic cardiomyopathy and normal cognitive development was diagnosed with an HRAS mutation c.173C>T (p.T58I), a milder variant of Costello syndrome affecting a highly conserved amino acid, threonine 58. Our analysis suggests that the p.G12 mutations slow GTP hydrolysis rendering HRAS unresponsive to GTPase activating proteins, and resulting in permanently active state. The p.T58I mutation likely affects binding of guanidine-nucleotide-exchange factors, thereby promoting the active state but also allowing for slow inactivation. Patients with the HRAS mutation c.173C>T (p.T58I) might go undiagnosed because of the milder phenotype compared with other mutations causing Costello syndrome. We expand the clinical and molecular picture of the rare HRAS mutation by reporting the first case in Europe and the fourth case in the literature. Our protein structure analysis offers insights into the mechanism of the mildly activating p.T58I mutation. © 2016 Wiley Periodicals, Inc.
Assuntos
Cardiomiopatia Hipertrófica/diagnóstico , Cardiomiopatia Hipertrófica/genética , Síndrome de Costello/diagnóstico , Síndrome de Costello/genética , Mutação , Fenótipo , Proteínas Proto-Oncogênicas p21(ras)/genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Alelos , Biomarcadores , Análise Mutacional de DNA , Ecocardiografia , Estudos de Associação Genética , Testes Genéticos , Genótipo , Humanos , Recém-Nascido , Masculino , Radiografia Torácica , Índice de Gravidade de DoençaRESUMO
The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations. The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.
RESUMO
Mutations in SUCLA2, encoding the ß-subunit of succinyl-CoA synthetase of Krebs cycle, are one cause of mitochondrial DNA depletion syndrome. Patients have been reported to have severe progressive childhood-onset encephalomyopathy, and methylmalonic aciduria, often leading to death in childhood. We studied two families, with children manifesting with slowly progressive mitochondrial encephalomyopathy, hearing impairment and transient methylmalonic aciduria, without mtDNA depletion. The other family also showed dominant inheritance of bilateral retinoblastoma, which coexisted with mitochondrial encephalomyopathy in one patient. We found a variant in SUCLA2 leading to Asp333Gly change, homozygous in one patient and compound heterozygous in one. The latter patient also carried a deletion of 13q14 of the other allele, discovered with molecular karyotyping. The deletion spanned both SUCLA2 and RB1 gene regions, leading to manifestation of both mitochondrial disease and retinoblastoma. We made a homology model for human succinyl-CoA synthetase and used it for structure-function analysis of all reported pathogenic mutations in SUCLA2. On the basis of our model, all previously described mutations were predicted to result in decreased amounts of incorrectly assembled protein or disruption of ADP phosphorylation, explaining the severe early lethal manifestations. However, the Asp333Gly change was predicted to reduce the activity of the otherwise functional enzyme. On the basis of our findings, SUCLA2 mutations should be analyzed in patients with slowly progressive encephalomyopathy, even in the absence of methylmalonic aciduria or mitochondrial DNA depletion. In addition, an encephalomyopathy in a patient with retinoblastoma suggests mutations affecting SUCLA2.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 13 , Heterozigoto , Encefalomiopatias Mitocondriais/genética , Mutação Puntual , Retinoblastoma/genética , Succinato-CoA Ligases/genética , Adolescente , Encéfalo/patologia , Hibridização Genômica Comparativa , Evolução Fatal , Frequência do Gene , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Encefalomiopatias Mitocondriais/complicações , Encefalomiopatias Mitocondriais/diagnóstico , Modelos Moleculares , Linhagem , Conformação Proteica , Retinoblastoma/complicações , Retinoblastoma/diagnóstico , Análise de Sequência de DNA , Relação Estrutura-Atividade , Succinato-CoA Ligases/químicaRESUMO
OBJECTIVE: We report novel defects of mitochondrial translation elongation factor Ts (EFTs), with high carrier frequency in Finland and expand the manifestations of this disease group from infantile cardiomyopathy to juvenile neuropathy/encephalopathy disorders. METHODS: DNA analysis, whole-exome analysis, protein biochemistry, and protein modeling. RESULTS: We used whole-exome sequencing to find the genetic cause of infantile-onset mitochondrial cardiomyopathy, progressing to juvenile-onset Leigh syndrome, neuropathy, and optic atrophy in 2 siblings. We found novel compound heterozygous mutations, c.944G>A [p.C315Y] and c.856C>T [p.Q286X], in the TSFM gene encoding mitochondrial EFTs. The same p.Q286X variant was found as compound heterozygous with a splice site change in a patient from a second family, with juvenile-onset optic atrophy, peripheral neuropathy, and ataxia. Our molecular modeling predicted the coding-region mutations to cause protein instability, which was experimentally confirmed in cultured patient cells, with mitochondrial translation defect and lacking EFTs. Only a single TSFM mutation has been previously described in different populations, leading to an infantile fatal multisystem disorder with cardiomyopathy. Sequence data from 35,000 Finnish population controls indicated that the heterozygous carrier frequency of p.Q286X change was exceptionally high in Finland, 1:80, but no homozygotes were found in the population, in our mitochondrial disease patient collection, or in an intrauterine fetal death material, suggesting early developmental lethality of the homozygotes. CONCLUSIONS: We show that in addition to early-onset cardiomyopathy, TSFM mutations should be considered in childhood and juvenile encephalopathies with optic and/or peripheral neuropathy, ataxia, or Leigh disease.
Assuntos
Ataxia/genética , DNA Mitocondrial/genética , Doença de Leigh/genética , Mitocôndrias/genética , Mutação/genética , Atrofia Óptica/genética , Fatores de Alongamento de Peptídeos/genética , Adolescente , Ataxia/diagnóstico , Feminino , Finlândia , Humanos , Doença de Leigh/diagnóstico , Atrofia Óptica/diagnóstico , Linhagem , Fenótipo , Adulto JovemRESUMO
Nutrient availability is the major regulator of life and reproduction, and a complex cellular signaling network has evolved to adapt organisms to fasting. These sensor pathways monitor cellular energy metabolism, especially mitochondrial ATP production and NAD(+)/NADH ratio, as major signals for nutritional state. We hypothesized that these signals would be modified by mitochondrial respiratory chain disease, because of inefficient NADH utilization and ATP production. Oral administration of nicotinamide riboside (NR), a vitamin B3 and NAD(+) precursor, was previously shown to boost NAD(+) levels in mice and to induce mitochondrial biogenesis. Here, we treated mitochondrial myopathy mice with NR. This vitamin effectively delayed early- and late-stage disease progression, by robustly inducing mitochondrial biogenesis in skeletal muscle and brown adipose tissue, preventing mitochondrial ultrastructure abnormalities and mtDNA deletion formation. NR further stimulated mitochondrial unfolded protein response, suggesting its protective role in mitochondrial disease. These results indicate that NR and strategies boosting NAD(+) levels are a promising treatment strategy for mitochondrial myopathy.
Assuntos
Mitocôndrias/efeitos dos fármacos , Miopatias Mitocondriais/tratamento farmacológico , Niacinamida/análogos & derivados , Complexo Vitamínico B/uso terapêutico , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/patologia , Animais , Metabolismo Energético/efeitos dos fármacos , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Miopatias Mitocondriais/metabolismo , Miopatias Mitocondriais/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , NAD/metabolismo , Niacinamida/uso terapêutico , Compostos de Piridínio , Sirtuína 1/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
BACKGROUND: The genetic complexity of infantile cardiomyopathies is remarkable, and the importance of mitochondrial translation defects as a causative factor is only starting to be recognised. We investigated the genetic basis for infantile onset recessive hypertrophic cardiomyopathy in two siblings. METHODS AND RESULTS: Analysis of respiratory chain enzymes revealed a combined deficiency of complexes I and IV in the heart and skeletal muscle. Exome sequencing uncovered a homozygous mutation (L156R) in MRPL44 of both siblings. MRPL44 encodes a protein in the large subunit of the mitochondrial ribosome and is suggested to locate in close proximity to the tunnel exit of the yeast mitochondrial ribosome. We found severely reduced MRPL44 levels in the patient's heart, skeletal muscle and fibroblasts suggesting that the missense mutation affected the protein stability. In patient fibroblasts, decreased MRPL44 affected assembly of the large ribosomal subunit and stability of 16S rRNA leading to complex IV deficiency. Despite this assembly defect, de novo mitochondrial translation was only mildly affected in fibroblasts suggesting that MRPL44 may have a function in the assembly/stability of nascent mitochondrial polypeptides exiting the ribosome. Retroviral expression of wild-type MRPL44 in patient fibroblasts rescued the large ribosome assembly defect and COX deficiency. CONCLUSIONS: These findings indicate that mitochondrial ribosomal subunit defects can generate tissue-specific manifestations, such as cardiomyopathy.