RESUMO
Inherited deficiency of thymidine phosphorylase (TP), encoded by TYMP, leads to a rare disease with multiple mitochondrial DNA (mtDNA) abnormalities, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the impact of TP deficiency on lysosomes remains unclear, which are important for mitochondrial quality control and nucleic acid metabolism. Muscle biopsy tissue and skin fibroblasts from MNGIE patients, patients with m.3243 A > G mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) and healthy controls (HC) were collected to perform mitochondrial and lysosomal functional analyses. In addition to mtDNA abnormalities, compared to controls distinctively reduced expression of LAMP1 and increased mitochondrial content were detected in the muscle tissue of MNGIE patients. Skin fibroblasts from MNGIE patients showed decreased expression of LAMP2, lowered lysosomal acidity, reduced enzyme activity and impaired protein degradation ability. TYMP knockout or TP inhibition in cells can also induce the similar lysosomal dysfunction. Using lysosome immunoprecipitation (Lyso- IP), increased mitochondrial proteins, decreased vesicular proteins and V-ATPase enzymes, and accumulation of various nucleosides were detected in lysosomes with TP deficiency. Treatment of cells with high concentrations of dThd and dUrd also triggers lysosomal dysfunction and disruption of mitochondrial homeostasis. Therefore, the results provided evidence that TP deficiency leads to nucleoside accumulation in lysosomes and lysosomal dysfunction, revealing the widespread disruption of organelles underlying MNGIE.
Assuntos
DNA Mitocondrial , Fibroblastos , Lisossomos , Mitocôndrias , Encefalomiopatias Mitocondriais , Nucleosídeos , Timidina Fosforilase , Humanos , Lisossomos/metabolismo , Timidina Fosforilase/metabolismo , Timidina Fosforilase/deficiência , Timidina Fosforilase/genética , Encefalomiopatias Mitocondriais/metabolismo , Encefalomiopatias Mitocondriais/patologia , Encefalomiopatias Mitocondriais/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Nucleosídeos/metabolismo , Pseudo-Obstrução Intestinal/metabolismo , Pseudo-Obstrução Intestinal/patologia , Pseudo-Obstrução Intestinal/enzimologia , Pseudo-Obstrução Intestinal/genética , Oftalmoplegia/metabolismo , Oftalmoplegia/patologia , Oftalmoplegia/congênito , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Masculino , Feminino , Pele/patologia , Pele/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismoAssuntos
Encéfalo/diagnóstico por imagem , Doença de Crohn/diagnóstico por imagem , Pseudo-Obstrução Intestinal/diagnóstico por imagem , Distrofia Muscular Oculofaríngea/diagnóstico por imagem , Oftalmoplegia/congênito , Substância Branca/diagnóstico por imagem , Doença de Crohn/genética , Diagnóstico Diferencial , Feminino , Humanos , Pseudo-Obstrução Intestinal/genética , Pessoa de Meia-Idade , Distrofia Muscular Oculofaríngea/genética , Oftalmoplegia/diagnóstico por imagem , Oftalmoplegia/genética , Timidina Fosforilase/deficiência , Timidina Fosforilase/genéticaRESUMO
BACKGROUND: MNGIE is a rare and fatal disease in which absence of the enzyme thymidine phosphorylase induces systemic accumulation of thymidine and deoxyuridine and secondary mitochondrial DNA alterations. Gastrointestinal (GI) symptoms are frequently reported in MNGIE patients, however, they are not resolved with the current treatment interventions. Recently, our understanding of the GI pathology has increased, which rationalizes the pursuit of more targeted therapeutic strategies. In particular, interstitial cells of Cajal (ICC) play key roles in GI physiology and are involved in the pathogenesis of the GI dysmotility. However, understanding of the triggers of ICC deficits in MNGIE is lacking. Herein, we review the current knowledge about the pathology of GI dysmotility in MNGIE, discuss potential mechanisms in relation to ICC loss/dysfunction, remark on the limited contribution of the current treatments, and propose intervention strategies to overcome ICC deficits. Finally, we address the advances and new research avenues offered by organoids and tissue engineering technologies, and propose schemes to implement to further our understanding of the GI pathology and utility in regenerative and personalized medicine in MNGIE. CONCLUSION: Interstitial cells of Cajal play key roles in the physiology of the gastrointestinal motility. Evaluation of their status in the GI dysmotility related to MNGIE would be valuable for diagnosis of MNGIE. Understanding the underlying pathological and molecular mechanisms affecting ICC is an asset for the development of targeted prevention and treatment strategies for the GI dysmotility related to MNGIE.
Assuntos
Gastroenteropatias/patologia , Células Intersticiais de Cajal/patologia , Timidina Fosforilase/deficiência , Feminino , Gastroenteropatias/metabolismo , Humanos , Células Intersticiais de Cajal/metabolismo , Pseudo-Obstrução Intestinal/metabolismo , Pseudo-Obstrução Intestinal/patologia , Masculino , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patologia , Mutação/genética , Oftalmoplegia/congênito , Timidina Fosforilase/genética , Timidina Fosforilase/metabolismoRESUMO
Mitochondrial neurogastrointestinal encephalomyopathy syndrome is a rare autosomal recessive multisystem disorder caused by nuclear TYMP gene mutations, which leads to deficiency in thymidine phosphorylase enzyme. This deficiency then leads to mitochondrial dysfunction, which causes the features characteristic of this syndrome, including severe muscle wasting, gastrointestinal dysmotility, leukoencephalopathy, peripheral neuropathy, and ophthalmoplegia. Here, we present a case series of 3 patients with mitochondrial neurogastrointestinal encephalomyopathy from Saudi Arabia who underwent allogeneic stem cell transplant at King Faisal Specialist Hospital (Riyadh, Saudi Arabia). Two patients died within the first year of transplant, and the third is still alive but without improvement in clinical features. Allogeneic hematopoietic stem cell transplant-related mortality appears to be high; this may at least be partially related to established end-organ effects with decreased performance status. Although allogeneic hematopoietic stem cell transplant clearly affects correction of genetic and biochemical defects in mitochondrial neurogastrointestinal encephalomyopathy, its ability to reverse or improve established clinical manifestations has not been proven.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Pseudo-Obstrução Intestinal/cirurgia , Distrofia Muscular Oculofaríngea/cirurgia , Adolescente , Adulto , Evolução Fatal , Predisposição Genética para Doença , Humanos , Pseudo-Obstrução Intestinal/diagnóstico , Pseudo-Obstrução Intestinal/enzimologia , Pseudo-Obstrução Intestinal/genética , Masculino , Distrofia Muscular Oculofaríngea/diagnóstico , Distrofia Muscular Oculofaríngea/enzimologia , Distrofia Muscular Oculofaríngea/genética , Mutação , Oftalmoplegia/congênito , Fenótipo , Timidina Fosforilase/deficiência , Timidina Fosforilase/genética , Fatores de Tempo , Transplante Homólogo , Resultado do TratamentoRESUMO
RATIONALE: Platelets contain abundant thymidine phosphorylase (TYMP), which is highly expressed in diseases with high risk of thrombosis, such as atherosclerosis and type II diabetes mellitus. OBJECTIVE: To test the hypothesis that TYMP participates in platelet signaling and promotes thrombosis. METHODS AND RESULTS: By using a ferric chloride (FeCl3)-induced carotid artery injury thrombosis model, we found time to blood flow cessation was significantly prolonged in Tymp(-/-) and Tymp(+/-) mice compared with wild-type mice. Bone marrow transplantation and platelet transfusion studies demonstrated that platelet TYMP was responsible for the antithrombotic phenomenon in the TYMP-deficient mice. Collagen-, collagen-related peptide-, adenosine diphosphate-, or thrombin-induced platelet aggregation were significantly attenuated in Tymp(+/-) and Tymp(-/-) platelets, and in wild type or human platelets pretreated with TYMP inhibitor KIN59. Tymp deficiency also significantly decreased agonist-induced P-selectin expression. TYMP contains an N-terminal SH3 domain-binding proline-rich motif and forms a complex with the tyrosine kinases Lyn, Fyn, and Yes in platelets. TYMP-associated Lyn was inactive in resting platelets, and TYMP trapped and diminished active Lyn after collagen stimulation. Tymp/Lyn double haploinsufficiency diminished the antithrombotic phenotype of Tymp(+/-) mice. TYMP deletion or inhibition of TYMP with KIN59 dramatically increased platelet-endothelial cell adhesion molecule 1 tyrosine phosphorylation and diminished collagen-related peptide- or collagen-induced AKT phosphorylation. In vivo administration of KIN59 significantly inhibited FeCl3-induced carotid artery thrombosis without affecting hemostasis. CONCLUSIONS: TYMP participates in multiple platelet signaling pathways and regulates platelet activation and thrombosis. Targeting TYMP might be a novel antiplatelet and antithrombosis therapy.
Assuntos
Plaquetas/enzimologia , Transdução de Sinais , Trombose/enzimologia , Timidina Fosforilase/metabolismo , Sequência de Aminoácidos , Animais , Plaquetas/efeitos dos fármacos , Transplante de Medula Óssea , Cloretos , Inibidores Enzimáticos/farmacologia , Compostos Férricos , Haploinsuficiência , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Fosforilação , Agregação Plaquetária , Inibidores da Agregação Plaquetária/farmacologia , Transfusão de Plaquetas , Proteínas Proto-Oncogênicas c-akt/sangue , Proteínas Proto-Oncogênicas c-fyn/sangue , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-yes/sangue , Selenoproteína P/sangue , Transdução de Sinais/efeitos dos fármacos , Trombose/sangue , Trombose/induzido quimicamente , Trombose/prevenção & controle , Timidina Fosforilase/antagonistas & inibidores , Timidina Fosforilase/sangue , Timidina Fosforilase/deficiência , Timidina Fosforilase/genética , Fatores de Tempo , Quinases da Família src/sangue , Quinases da Família src/genéticaRESUMO
Mycoplasma pneumoniae (Mpn) is a human pathogen causing acute respiratory diseases and accounts for approximately 30% cases of community-acquired pneumonia. Co-infection with Mycoplasmas compromises the efficacy of anticancer and antiviral nucleoside analog-based drugs due to the presence of Mycoplasma thymidine phosphorylase (TP). In this study, a TP-deficient strain of Mpn was generated in order to study the effect of Mpn TP in the metabolism of nucleoside analogs. Deficiency in TP activity led to increased uptake and incorporation of radiolabeled deoxyuridine and uracil but thymidine uptake was not affected. The activities of enzymes in the salvage of thymidine and deoxyuridine, e.g., thymidine kinase and uracil phosphoribosyltransferase were upregulated in the TP-deficient mutant, which may explain the increased uptake of deoxyuridine and uracil. Thirty FDA-approved anticancer and antiviral nucleoside and nucleobase analogs were used to screen their inhibitory activity toward the TP mutant and the wild type strain. Seven analogs were found to inhibit strongly the growth of both wild type and TP mutant. Differences in the inhibitory effect of several purine analogs between the two strains were observed. Further study is needed in order to understand the mechanism of inhibition caused by these analogs. Our results indicated that TP is not an essential gene for Mpn survival and TP deficiency affects other enzymes in Mpn nucleotide metabolism, and suggested that Mycoplasma nucleotide biosynthesis pathway enzymes are potential targets for future development of antibiotics.
Assuntos
Mycoplasma pneumoniae/enzimologia , Timidina Fosforilase/metabolismo , Antineoplásicos/farmacologia , Antivirais/farmacologia , Desoxiuridina/metabolismo , Mutação , Mycoplasma pneumoniae/efeitos dos fármacos , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/crescimento & desenvolvimento , Timidina/metabolismo , Timidina Fosforilase/deficiência , Timidina Fosforilase/genéticaRESUMO
TAS-102 (trifluorothymidine [TFT] and thymidine phosphorylase inhibitor [TPI] in a molar ratio of 1:0.5) has activity in 5-fluorouracil resistant colon cancer. TPI is added to increase TFT's bioavailability. TFT has a dual mechanism of action by inhibiting thymidylate synthase and by its incorporation into DNA. Interesting radiosensitizing effects of TPI were recently reported. The aim of our study was to determine whether TP expression would affect radiosensitivity and to characterize the effect of TPI. Two bladder cancer cell lines RT112 (TP negative) and RT112/TP (TP overexpression) were tested for drug sensitivity and radiosensitivity (clonogenic assay), with and without TFT and/or TPI. Expression of γ H2AX was used as marker for DNA damage. RT112 cells were not more sensitive to TFT then RT112/TP cells. TPI alone did not inhibit cell growth of RT112 even at 100 µM, but inhibited that of RT112/TP by 27%. In both RT112 and RT112/TP cells 10 µM TPI did not or slightly affect radiosensitivity, but 100 µM TPI alone enhanced the radiation response (p<.05). TFT alone at 1 µM and in combination with 10 µM TPI did not affect the radiation response of both cell lines. TPI alone induced expression of ÏH2AX, which was increased in combination with radiation. In conclusion, TPI enhanced radiosensitivity at high concentrations, independent of TP expression, while TFT and TPI at a low concentration did not affect the radiosensitivity of RT112 and RT112/TP cell lines.
Assuntos
Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Timidina Fosforilase/antagonistas & inibidores , Timidina Fosforilase/metabolismo , Trifluridina/farmacologia , Uracila/análogos & derivados , Neoplasias da Bexiga Urinária/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Dano ao DNA , Combinação de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Pirrolidinas , Timidina Fosforilase/deficiência , Timina , Uracila/farmacologiaRESUMO
Balanced pools of deoxyribonucleoside triphosphate precursors are required for DNA replication, and alterations of this balance are relevant to human mitochondrial diseases including mitochondrial neurogastrointestinal encephalopathy. In this disease, autosomal recessive TYMP mutations cause severe reductions of thymidine phosphorylase activity; marked elevations of the pyrimidine nucleosides thymidine and deoxyuridine in plasma and tissues, and somatic multiple deletions, depletion and site-specific point mutations of mitochondrial DNA. Thymidine phosphorylase and uridine phosphorylase double knockout mice recapitulated several features of these patients including thymidine phosphorylase activity deficiency, elevated thymidine and deoxyuridine in tissues, mitochondrial DNA depletion, respiratory chain defects and white matter changes. However, in contrast to patients with this disease, mutant mice showed mitochondrial alterations only in the brain. To test the hypothesis that elevated levels of nucleotides cause unbalanced deoxyribonucleoside triphosphate pools and, in turn, pathogenic mitochondrial DNA instability, we have stressed double knockout mice with exogenous thymidine and deoxyuridine, and assessed clinical, neuroradiological, histological, molecular, and biochemical consequences. Mutant mice treated with exogenous thymidine and deoxyuridine showed reduced survival, body weight, and muscle strength, relative to untreated animals. Moreover, in treated mutants, leukoencephalopathy, a hallmark of the disease, was enhanced and the small intestine showed a reduction of smooth muscle cells and increased fibrosis. Levels of mitochondrial DNA were depleted not only in the brain but also in the small intestine, and deoxyribonucleoside triphosphate imbalance was observed in the brain. The relative proportion, rather than the absolute amount of deoxyribonucleoside triphosphate, was critical for mitochondrial DNA maintenance. Thus, our results demonstrate that stress of exogenous pyrimidine nucleosides enhances the mitochondrial phenotype of our knockout mice. Our mouse studies provide insights into the pathogenic role of thymidine and deoxyuridine imbalance in mitochondrial neurogastrointestinal encephalopathy and an excellent model to study new therapeutic approaches.
Assuntos
Desoxirribonucleosídeos/efeitos adversos , Pseudo-Obstrução Intestinal/induzido quimicamente , Pseudo-Obstrução Intestinal/genética , Encefalomiopatias Mitocondriais/induzido quimicamente , Encefalomiopatias Mitocondriais/genética , Fatores Etários , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Encéfalo/patologia , Desoxirribonucleosídeos/metabolismo , Modelos Animais de Doenças , Pseudo-Obstrução Intestinal/mortalidade , Pseudo-Obstrução Intestinal/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Mitocondriais/etiologia , Doenças Mitocondriais/genética , Encefalomiopatias Mitocondriais/mortalidade , Encefalomiopatias Mitocondriais/fisiopatologia , Atividade Motora/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Força Muscular/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Oculofaríngea , Oftalmoplegia/congênito , Transtornos Psicomotores/etiologia , Transtornos Psicomotores/genética , Succinato Desidrogenase/metabolismo , Timidina/efeitos adversos , Timidina/metabolismo , Timidina Fosforilase/deficiência , Uridina Fosforilase/deficiênciaRESUMO
BACKGROUND: Although causative mutations have been identified for numerous mitochondrial disorders, few disease-modifying treatments are available. Two examples of treatable mitochondrial disorders are coenzyme Q(10) (CoQ(10) or ubiquinone) deficiency and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). SCOPE OF REVIEW: Here, we describe clinical and molecular features of CoQ(10) deficiencies and MNGIE and explain how understanding their pathomechanisms have led to rationale therapies. Primary CoQ(10) deficiencies, due to mutations in genes required for ubiquinone biosynthesis, and secondary deficiencies, caused by genetic defects not directly related to CoQ(10) biosynthesis, often improve with CoQ(10) supplementation. In vitro and in vivo studies of CoQ(10) deficiencies have revealed biochemical alterations that may account for phenotypic differences among patients and variable responses to therapy. In contrast to the heterogeneous CoQ(10) deficiencies, MNGIE is a single autosomal recessive disease due to mutations in the TYMP gene encoding thymidine phosphorylase (TP). In MNGIE, loss of TP activity causes toxic accumulations of the nucleosides thymidine and deoxyuridine that are incorporated by the mitochondrial pyrimidine salvage pathway and cause deoxynucleoside triphosphate pool imbalances, which, in turn cause mtDNA instability. Allogeneic hematopoetic stem cell transplantation to restore TP activity and eliminate toxic metabolites is a promising therapy for MNGIE. MAJOR CONCLUSIONS: CoQ(10) deficiencies and MNGIE demonstrate the feasibility of treating specific mitochondrial disorders through replacement of deficient metabolites or via elimination of excessive toxic molecules. GENERAL SIGNIFICANCE: Studies of CoQ(10) deficiencies and MNGIE illustrate how understanding the pathogenic mechanisms of mitochondrial diseases can lead to meaningful therapies. This article is part of a Special Issue entitled: Biochemistry of Mitochondria, Life and Intervention 2010.
Assuntos
Doenças Mitocondriais/terapia , Encefalomiopatias Mitocondriais/terapia , Timidina Fosforilase/deficiência , Ubiquinona/análogos & derivados , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/metabolismo , Ubiquinona/deficiênciaRESUMO
Deficiency of the cytosolic enzyme thymidine phosphorylase (TP) causes a multisystem disorder called mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) syndrome. Clinical symptoms are gastrointestinal dysfunction, muscle involvement and neurological deterioration. TP deficiency is biochemically characterised by accumulation of thymidine and deoxyuridine in body fluids and compromised mitochondrial deoxyribose nucleic acid (mtDNA) integrity (depletion and multiple deletions). In this report we describe a patient with the clinical and biochemical features related to the end stage of the disease. Home parenteral nutrition had started to improve the clinical condition and preparations were initiated for stem cell transplantation (SCT) as a last resort treatment. Unfortunately, the patient died during the induction phase of SCT. This report shows that TP deficiency is a severe clinical condition with a broad spectrum of affected tissues. TP deficiency can be easily determined by the measurement of pyrimidine metabolites in body fluids and TP activity in peripheral blood leucocytes. Early detection and treatment may prevent the progress of the clinical symptoms and, therefore, should be considered for inclusion in newborn screening programmes.
Assuntos
Pseudo-Obstrução Intestinal/enzimologia , Encefalomiopatias Mitocondriais/enzimologia , Timidina Fosforilase/deficiência , Adolescente , Adulto , Biomarcadores/análise , Análise Mutacional de DNA , Progressão da Doença , Evolução Fatal , Feminino , Predisposição Genética para Doença , Testes Genéticos , Humanos , Pseudo-Obstrução Intestinal/diagnóstico , Pseudo-Obstrução Intestinal/genética , Pseudo-Obstrução Intestinal/terapia , Masculino , Encefalomiopatias Mitocondriais/diagnóstico , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/terapia , Distrofia Muscular Oculofaríngea , Oftalmoplegia/congênito , Nutrição Parenteral no Domicílio , Linhagem , Fenótipo , Prognóstico , Índice de Gravidade de Doença , Transplante de Células-Tronco , Timidina Fosforilase/genética , Fatores de TempoRESUMO
Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, ptosis, ophthalmoplegia, and leukoencephalopathy with early onset and severe prognosis. Mutations in the TYMP/ECGF1 gene cause a loss of thymidine phosphorylase catalytic activity, disrupting the homeostasis of intramitochondrial nucleotide pool. We report a woman with a very late onset of MNGIE, lacking peripheral neuropathy. Thymidine phosphorylase activity was markedly reduced in cultured fibroblasts, but only mildly reduced in buffy coat, where the defect is usually detected, and plasma thymidine was mildly increased compared to typical MNGIE patients. TYMP/ECGF1 analysis detected two heterozygous mutations, including a novel missense mutation. These findings indicate that a partial loss of thymidine phosphorylase activity may induce a late-onset and incomplete MNGIE phenotype.
Assuntos
Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/fisiopatologia , Timidina Fosforilase/deficiência , Idade de Início , Encéfalo/patologia , Encéfalo/fisiopatologia , Células Cultivadas , Feminino , Fibroblastos/enzimologia , Humanos , Pessoa de Meia-Idade , Encefalomiopatias Mitocondriais/sangue , Mutação , Mutação de Sentido Incorreto , Nervos Periféricos/fisiopatologia , Fenótipo , Timidina/sangue , Timidina Fosforilase/genéticaRESUMO
Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability.
Assuntos
DNA Mitocondrial/química , Desoxirribonucleotídeos/metabolismo , Instabilidade Genômica , Erros Inatos do Metabolismo da Purina-Pirimidina/metabolismo , Timidina Fosforilase/deficiência , Uridina Fosforilase/metabolismo , Animais , Encéfalo/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Erros Inatos do Metabolismo da Purina-Pirimidina/genética , Timidina Fosforilase/genética , Uridina Fosforilase/genéticaRESUMO
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy and leukoencephalopathy. The disease is due to a thymidine phosphorylase defect. This enzyme catalyses the phosphorolysis of thymidine to thymine and deoxyribose 1-phosphate. For this reason, increased levels of thymidine in plasma and urine are found in MNGIE patients. Haemodialysis can reduce circulating plasma thymidine levels and can be beneficial in some MNGIE patients. We developed a fast analytical method based on HPLC-ESI-MS/MS capable of identifying pyrimidine nucleotides (thymine, cytosine, uracil) and nucleosides (thymidine, citidine, uridine) in plasma and urine after direct dilution of the samples without pre-treatment. In the patient studied, we observed a significant reduction of plasmatic and urinary thymidine levels during and after dialysis. However, we noted a progressive reduction of the initial thymidine level after some dialytic trials. This method will be useful not only for thymidine level follow-up during dialysis in MNGIE patients but also for the improvement of the diagnosis or diagnostic suspect in other pyrimidine defects such as dihydropyrimidine dehydrogenase deficiency, dihydropyrimidinase deficiency and ureidopropionase deficiency.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Encefalomiopatias Mitocondriais/sangue , Encefalomiopatias Mitocondriais/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Timidina , Adulto , Evolução Fatal , Feminino , Humanos , Encefalomiopatias Mitocondriais/terapia , Diálise Renal , Timidina/análise , Timidina/sangue , Timidina/urina , Timidina Fosforilase/deficiênciaRESUMO
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TP). The disease is characterized clinically by impaired eye movements, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy, and leukoencephalopathy. Molecular genetic studies of MNGIE patients' tissues have revealed multiple deletions, depletion, and site-specific point mutations of mitochondrial DNA. TP is a cytosolic enzyme required for nucleoside homeostasis. In MNGIE, TP activity is severely reduced and consequently levels of thymidine and deoxyuridine in plasma are dramatically elevated. We have hypothesized that the increased levels of intracellular thymidine and deoxyuridine cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities. MNGIE was the first molecularly characterized genetic disorder caused by abnormal mitochondrial nucleoside/nucleotide metabolism. Future studies are likely to reveal further insight into this expanding group of diseases.
Assuntos
Encefalopatias/genética , Doenças Mitocondriais/genética , Doenças Musculares/genética , Timidina Fosforilase/deficiência , Timidina Fosforilase/genética , Southern Blotting , Cromatografia Líquida de Alta Pressão , Genes Recessivos , Ligação Genética , Heterozigoto , Humanos , Doenças Mitocondriais/enzimologia , Modelos Biológicos , Mutação , Mutação Puntual , Espectrofotometria , Timidina/metabolismoRESUMO
Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive multisystemic disorder caused by thymidine phosphorylase deficiency. Whereas the pathomechanism of the secondary mitochondrial dysfunction has been extensively studied, that of the leukoencephalopathy has not been elucidated. We hypothesized that the white matter hyperintensities on T2-weighted magnetic resonance images reflect disturbance of blood-brain barrier function. Albumin immunohistochemistry disclosed quantitative (p < 0.01) and qualitative differences between the mitochondrial neurogastrointestinal encephalomyopathy and control brains, indicating that loss of thymidine phosphorylase function impairs the integrity of the blood-brain barrier.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/enzimologia , Permeabilidade Capilar , Timidina Fosforilase/deficiência , Adolescente , Adulto , Barreira Hematoencefálica/patologia , Encéfalo/patologia , HumanosRESUMO
We report five patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) who had demyelinating peripheral neuropathy. The MNGIE neuropathy had clinical and electrodiagnostic features typical of acquired, rather than inherited, etiologies. In fact, three patients were actually treated for chronic inflammatory demyelinating polyneuropathy (CIDP). We discuss findings that may help distinguish patients with MNGIE from those with CIDP.
Assuntos
Erros de Diagnóstico/prevenção & controle , Encefalomiopatias Mitocondriais/diagnóstico , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/diagnóstico , Adjuvantes Imunológicos/uso terapêutico , Adolescente , Corticosteroides/uso terapêutico , Adulto , Axônios/metabolismo , Axônios/patologia , Diagnóstico Diferencial , Eletrodiagnóstico , Feminino , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Masculino , Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/fisiopatologia , Fibras Nervosas Mielinizadas/metabolismo , Fibras Nervosas Mielinizadas/patologia , Condução Nervosa/genética , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Nervos Periféricos/fisiopatologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/fisiopatologia , Timidina Fosforilase/deficiência , Timidina Fosforilase/genéticaRESUMO
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5'-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations.
Assuntos
DNA Mitocondrial/genética , Encefalomiopatias Mitocondriais/enzimologia , Encefalomiopatias Mitocondriais/genética , Mutação Puntual , Timidina Fosforilase/deficiência , Timidina Fosforilase/genética , Sequência de Bases , Células Cultivadas , Análise Mutacional de DNA , Desoxiuridina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Gastroenteropatias/enzimologia , Gastroenteropatias/genética , Humanos , Modelos Genéticos , Polimorfismo de Fragmento de Restrição , Deleção de SequênciaRESUMO
Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.
Assuntos
Desoxiuridina/sangue , Timidina Fosforilase/deficiência , Sequência de Bases , Bioquímica/métodos , Relação Dose-Resposta a Droga , Humanos , Cinética , Dados de Sequência Molecular , Mutação , Nucleosídeos/química , Mutação Puntual , Valores de Referência , Especificidade por Substrato , Timidina/metabolismo , Fatores de TempoRESUMO
Thymidine phosphorylase (TP) regulates intracellular and plasma thymidine levels. TP deficiency is hypothesized to (i) increase levels of thymidine in plasma, (ii) lead to mitochondrial DNA alterations, and (iii) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). In order to elucidate the physiological roles of TP, we generated mice deficient in the TP gene. Although TP activity in the liver was inhibited in these mice, it was fully maintained in the small intestine. Murine uridine phosphorylase (UP), unlike human UP, cleaves thymidine, as well as uridine. We therefore generated TP-UP double-knockout (TP(-/-) UP(-/-)) mice. TP activities were inhibited in TP(-/-) UP(-/-) mice, and the level of thymidine in the plasma of TP(-/-) UP(-/-) mice was higher than for TP(-/-) mice. Unexpectedly, we could not observe alterations of mitochondrial DNA or pathological changes in the muscles of the TP(-/-) UP(-/-) mice, even when these mice were fed thymidine for 7 months. However, we did find hyperintense lesions on magnetic resonance T(2) maps in the brain and axonal edema by electron microscopic study of the brain in TP(-/-) UP(-/-) mice. These findings suggested that the inhibition of TP activity caused the elevation of pyrimidine levels in plasma and consequent axonal swelling in the brains of mice. Since lesions in the brain do not appear to be due to mitochondrial alterations and pathological changes in the muscle were not found, this model will provide further insights into the causes of MNGIE.