RESUMO
Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Homocistinúria/genética , Fator C1 de Célula Hospedeira/genética , Oxirredutases/genética , Proteínas Repressoras/genética , Ribossomos/genética , Deficiência de Vitamina B 12/genética , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Animais , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Homocistinúria/metabolismo , Homocistinúria/patologia , Fator C1 de Célula Hospedeira/deficiência , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Biogênese de Organelas , Oxirredutases/deficiência , Biossíntese de Proteínas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Repressoras/deficiência , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Ribossomos/patologia , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologiaRESUMO
BACKGROUND: Homocystinuria is an autosomal recessive metabolic disorder occurring due to the defects in cystathionine-ß-synthase enzyme. The study was carried out to investigate a Pakistani family presenting bilateral congenital cataract with symptoms of classical homocystinuria at LRBT Free Eye Hospital, Lahore, Pakistan. METHODS: Three affected individuals of the family presented skeletal deformations, intellectual disability, speech delay, and myopia with bilateral congenital cataract. Genetic analysis on DNA samples from affected individuals was done through whole exome sequencing to identify underlying genetic variant causing disease phenotypes in the family. In silico analysis was done to predict the effect of variation on the structure of mutant protein. RESULTS: A missense allelic variant (NM_000071.3: c.253G>A) of the CBS gene was revealed which may affect the catalytic activity of the substituted (NP_000062.1: p.G85R) protein by disrupting the folding of the enzymatic protein. High levels of homocysteine were observed in the plasma of affected individuals. This is the first report of this genetic variant from Pakistan causing homocystinuria and congenital cataract in association. CONCLUSION: This variant was reported first time in association with congenital cataract instead of ectopia lentis. Congenital cataract was developed secondarily in these patients and provided a clue for the early diagnosis of metabolic disorders like homocystinuria to prevent further complications and morbidity.
Assuntos
Catarata/genética , Cistationina beta-Sintase/genética , Homocistinúria/genética , Fenótipo , Catarata/patologia , Criança , Cistationina beta-Sintase/química , Feminino , Homocistinúria/patologia , Humanos , Lactente , Masculino , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , LinhagemRESUMO
Cobalamin J disease (CblJ) is an ultra-rare autosomal recessive disorder of intracellular cobalamin metabolism associated with combined methylmalonic acidemia and homocystinuria. It is caused by pathogenic variants in ABCD4, which encodes an ATP-binding cassette (ABC) transporter that affects the lysosomal release of cobalamin (Cbl) into the cytoplasm. Only six cases of CblJ have been reported in the literature. Described clinical features include feeding difficulties, failure to thrive, hypotonia, seizures, developmental delay, and hematological abnormalities. Information on clinical outcomes is extremely limited, and no cases of presymptomatic diagnosis have been reported. We describe a now 17-month-old male with CblJ detected by newborn screening and confirmed by biochemical, molecular, and complementation studies. With early detection and initiation of treatment, this patient has remained asymptomatic with normal growth parameters and neurodevelopmental function. To the best of our knowledge, this report represents the first asymptomatic and neurotypical patient with CblJ.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico , Erros Inatos do Metabolismo dos Aminoácidos/genética , Deficiência de Vitamina B 12/diagnóstico , Vitamina B 12/genética , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Feminino , Predisposição Genética para Doença , Homocistinúria/diagnóstico , Homocistinúria/genética , Homocistinúria/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Ácido Metilmalônico/metabolismo , Mutação/genética , Triagem Neonatal , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/genética , Deficiência de Vitamina B 12/patologiaRESUMO
We report a patient with homocystinuria and hyperoxaluria who was cured of homocystinuria-related disease following liver transplant. The patient was diagnosed with homocystinuria as a newborn and was treated with dietary modifications and supplements. At 22 months, he passed a calcium oxalate stone and was found to have numerous bilateral kidney stones. Genetic testing confirmed primary hyperoxaluria, type 1. He underwent preemptive liver transplant at age four to treat primary hyperoxaluria. Following transplant, his serum methionine and homocysteine levels normalized, thus, demonstrating resolution of homocystinuria. Methionine and homocysteine levels remained normal 6 years later. Homocystinuria is associated with ophthalmologic, skeletal, neurologic, and thromboembolic complications. As cystathionine beta-synthase resides in the liver, transplant was hypothesized to be an effective treatment. Primary hyperoxaluria generally progresses to chronic kidney disease and is treated with combined kidney-liver transplant at the time of end stage kidney disease. Given this patient's dual diagnoses, we proceeded with preemptive liver transplantation. Three prior cases of patients with homocystinuria treated with liver transplantation have been reported. In all cases, transplant resolved metabolic effects. However, our case represents a pediatric patient without disease-related complications prior to transplant. This case supports liver-targeted gene therapies as an effective treatment for homocystinuria.
Assuntos
Cistationina beta-Sintase/genética , Homocistinúria/genética , Homocistinúria/terapia , Transplante de Fígado , Cistationina beta-Sintase/deficiência , Feminino , Homocisteína/sangue , Homocistinúria/sangue , Homocistinúria/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Metionina/sangue , Triagem Neonatal , PediatriaRESUMO
Cystathionine beta-synthase deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. Our knowledge of the metabolic changes induced in HCU are based almost exclusively on data derived from plasma. In the present study, we present a comprehensive analysis on the effects of HCU upon the hepatic metabolites and enzyme expression levels of the methionine-folate cycles in a mouse model of HCU. HCU induced a 10-fold increase in hepatic total homocysteine and in contrast to plasma, this metabolite was only lowered by approximately 20% by betaine treatment indicating that this toxic metabolite remains unacceptably elevated. Hepatic methionine, S-adenosylmethionine, S-adenosylhomocysteine, N-acetlymethionine, N-formylmethionine, methionine sulfoxide, S-methylcysteine, serine, N-acetylserine, taurocyamine and N-acetyltaurine levels were also significantly increased by HCU while cysteine, N-acetylcysteine and hypotaurine were all significantly decreased. In terms of polyamine metabolism, HCU significantly decreased spermine and spermidine levels while increasing 5'-methylthioadenosine. Betaine treatment restored normal spermine and spermidine levels but further increased 5'-methylthioadenosine. HCU induced a 2-fold induction in expression of both S-adenosylhomocysteine hydrolase and methylenetetrahydrofolate reductase. Induction of this latter enzyme was accompanied by a 10-fold accumulation of its product, 5-methyl-tetrahydrofolate, with the potential to significantly perturb onecarbon metabolism. Expression of the cytoplasmic isoform of serine hydroxymethyltransferase was unaffected by HCU but the mitochondrial isoform was repressed indicating differential regulation of onecarbon metabolism in different sub-cellular compartments. All HCU-induced changes in enzyme expression were completely reversed by either betaine or taurine treatment. Collectively, our data show significant alterations of polyamine, folate and methionine cycle metabolism in HCU hepatic tissues that in some cases, differ significantly from those observed in plasma, and have the potential to contribute to multiple aspects of pathogenesis.
Assuntos
Cistationina beta-Sintase/genética , Homocistinúria/metabolismo , Fígado/metabolismo , Metionina/metabolismo , Adenosil-Homocisteinase/genética , Animais , Betaína/farmacologia , Cistationina beta-Sintase/metabolismo , Modelos Animais de Doenças , Ácido Fólico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicina Hidroximetiltransferase/genética , Homocisteína/sangue , Homocisteína/metabolismo , Homocistinúria/tratamento farmacológico , Homocistinúria/genética , Homocistinúria/patologia , Humanos , Fígado/enzimologia , Metionina/análogos & derivados , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Camundongos , Poliaminas/metabolismoRESUMO
5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency usually presents as a severe neonatal disease. This study aimed to characterize natural history, biological and molecular data, and response to treatment of patients with late-onset MTHFR deficiency. The patients were identified through the European Network and Registry for Homocystinuria and Methylation Defects and the Adult group of the French Society for Inherited Metabolic Diseases; data were retrospectively colleted. To identify juvenile to adult-onset forms of the disease, we included patients with a diagnosis established after the age of 10 years. We included 14 patients (median age at diagnosis: 32 years; range: 11-54). At onset (median age: 20 years; range 9-38), they presented with walking difficulties (n = 8), cognitive decline (n = 3) and/or seizures (n = 3), sometimes associated with mild mental retardation (n = 6). During the disease course, symptoms were almost exclusively neurological with cognitive dysfunction (93%), gait disorders (86%), epilepsy (71%), psychiatric symptoms (57%), polyneuropathy (43%), and visual deficit (43%). Mean diagnostic delay was 14 years. Vascular events were observed in 28% and obesity in 36% of the patients. One patient remained asymptomatic at the age of 55 years. Upon treatment, median total homocysteine decreased (from 183 µmol/L, range 69-266, to 90 µmol/L, range 20-142) and symptoms improved (n = 9) or stabilized (n = 4). Missense pathogenic variants in the C-terminal regulatory domain of the protein were over-represented compared to early-onset cases. Residual MTHFR enzymatic activity in skin fibroblasts (n = 4) was rather high (17%-58%). This series of patients with late-onset MTHFR deficiency underlines the still unmet need of a prompt diagnosis of this treatable disease.
Assuntos
Homocistinúria/diagnóstico , Homocistinúria/patologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/patologia , Adolescente , Adulto , Idade de Início , Criança , Diagnóstico Tardio , Epilepsia/diagnóstico , Epilepsia/patologia , Feminino , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Masculino , Pessoa de Meia-Idade , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/patologia , Estudos Retrospectivos , Convulsões/diagnóstico , Convulsões/patologia , Adulto JovemRESUMO
Combined methylmalonic acidemia and homocystinuria, cblC type, is the most common inherited disorder of cobalamin metabolism and is characterized by severe fetal developmental defects primarily impacting the central nervous system, hematopoietic system, and heart. CblC was previously shown to be due to mutations in the MMACHC gene, which encodes a protein thought to function in intracellular cobalamin trafficking and biosynthesis of adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl). These coenzymes are required for the production of succinyl-CoA and methionine, respectively. However, it is currently unclear whether additional roles for MMACHC exist outside of cobalamin metabolism. Furthermore, due to a lack of sufficient animal models, the exact pathophysiology of cblC remains unknown. Here, we report the generation and characterization of two new mouse models to study the role of MMACHC in vivo. CRISPR/Cas9 genome editing was used to develop a Mmachc floxed allele (Mmachcflox/flox), which we validated as a conditional null. For a gain-of-function approach, we generated a transgenic mouse line that over-expresses functional Mmachc (Mmachc-OE+/tg) capable of rescuing Mmachc homozygous mutant lethality. Surprisingly, our data also suggest that these mice may exhibit a partially penetrant maternal-effect rescue, which might have implications for in utero therapeutic interventions to treat cblC. Both the Mmachcflox/flox and Mmachc-OE+/tg mouse models will be valuable resources for understanding the biological roles of MMACHC in a variety of tissue contexts and allow for deeper understanding of the pathophysiology of cblC.
Assuntos
Homocistinúria , Oxirredutases , Deficiência de Vitamina B 12/congênito , Animais , Modelos Animais de Doenças , Homocistinúria/genética , Homocistinúria/metabolismo , Homocistinúria/patologia , Homocistinúria/fisiopatologia , Camundongos , Camundongos Transgênicos , Oxirredutases/genética , Oxirredutases/metabolismo , Deficiência de Vitamina B 12/genética , Deficiência de Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologia , Deficiência de Vitamina B 12/fisiopatologiaRESUMO
Classic homocystinuria is due to deficiency of cystathionine beta-synthase (CBS), a pyridoxine-dependent enzyme that, depending on the molecular variants, may be co-factor responsive. Elevated methionine is often used as the primary analyte to detect CBS deficiency (CBSD) on newborn screening (NBS), but is limited by increased detection of other biochemical disorders with less clear clinical significance such as methionine aminotransferase (MAT) I/III heterozygotes. Our state has implemented a two-tier NBS algorithm for CBSD that successfully reduced the number of MATI/III heterozygotes, yet effectively detected a mild, co-factor responsive form of CBSD. After initial diagnosis, newborns with CBSD often undergo a pyridoxine challenge with high-dose pyridoxine to determine responsiveness. Here we describe our NBS-identified patient with a mild form of pyridoxine responsive CBSD who developed respiratory failure and rhabdomyolysis consistent with pyridoxine toxicity during a pyridoxine challenge. This case highlights the need for weight-based dosing and duration recommendations for pyridoxine challenge in neonates.
Assuntos
Cistationina beta-Sintase/deficiência , Cistationina beta-Sintase/genética , Homocistinúria/tratamento farmacológico , Triagem Neonatal/métodos , Piridoxina/efeitos adversos , Insuficiência Respiratória/patologia , Rabdomiólise/patologia , Relação Dose-Resposta a Droga , Feminino , Homocistinúria/genética , Homocistinúria/patologia , Humanos , Recém-Nascido , Prognóstico , Piridoxina/administração & dosagem , Insuficiência Respiratória/induzido quimicamente , Rabdomiólise/induzido quimicamente , Complexo Vitamínico B/administração & dosagem , Complexo Vitamínico B/efeitos adversosRESUMO
Cobalamin C (cblC) deficiency, the most common inborn error of intracellular cobalamin metabolism, is caused by mutations in MMACHC, a gene responsible for the processing and intracellular trafficking of vitamin B12. This recessive disorder is characterized by a failure to metabolize cobalamin into adenosyl- and methylcobalamin, which results in the biochemical perturbations of methylmalonic acidemia, hyperhomocysteinemia and hypomethioninemia caused by the impaired activity of the downstream enzymes, methylmalonyl-CoA mutase and methionine synthase. Cobalamin C deficiency can be accompanied by a wide spectrum of clinical manifestations, including progressive blindness, and, in mice, manifests with very early embryonic lethality. Because zebrafish harbor a full complement of cobalamin metabolic enzymes, we used genome editing to study the loss of mmachc function and to develop the first viable animal model of cblC deficiency. mmachc mutants survived the embryonic period but perished in early juvenile life. The mutants displayed the metabolic and clinical features of cblC deficiency including methylmalonic acidemia, severe growth retardation and lethality. Morphologic and metabolic parameters improved when the mutants were raised in water supplemented with small molecules used to treat patients, including hydroxocobalamin, methylcobalamin, methionine and betaine. Furthermore, mmachc mutants bred to express rod and/or cone fluorescent reporters, manifested a retinopathy and thin optic nerves (ON). Expression analysis using whole eye mRNA revealed the dysregulation of genes involved in phototransduction and cholesterol metabolism. Zebrafish with mmachc deficiency recapitulate the several of the phenotypic and biochemical features of the human disorder, including ocular pathology, and show a response to established treatments.
Assuntos
Proteínas de Transporte/genética , Morfogênese/genética , Deficiência de Vitamina B 12/genética , Vitamina B 12/genética , Proteínas de Peixe-Zebra/genética , Animais , Homocistinúria/genética , Homocistinúria/patologia , Humanos , Camundongos , Mutação/genética , Nervo Óptico/crescimento & desenvolvimento , Nervo Óptico/patologia , Oxirredutases/genética , Retina/crescimento & desenvolvimento , Retina/metabolismo , Vitamina B 12/análogos & derivados , Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/metabolismo , Deficiência de Vitamina B 12/patologia , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
Methylene tetrahydrofolate reductase (MTHFR) is a flavoprotein, involved in one-carbon pathway and is responsible for folate and homocysteine metabolism. Regulation of MTHFR is pivotal for maintaining the cellular concentrations of methionine and SAM (S-adenosyl methionine) which are essential for the synthesis of nucleotides and amino acids, respectively. Therefore, mutations in MTHFR leads to its dysfunction resulting in conditions like homocystinuria, cardiovascular diseases, and neural tube defects in infants. Among these conditions, homocystinuria has been highly explored, as it manifests ocular disorders, cognitive disorders and skeletal abnormalities. Hence, in this study, we intend to explore the mutational landscape of human MTHFR isoform-1 (h.MTHFR-1) to decipher the most pathogenic variants pertaining to homocystinuria. Thus, a multilevel stringent prioritization of non-synonymous mutations in h.MTHFR-1 by integrative machine learning approaches was implemented to delineate highly deleterious variants based on its pathogenicity, impact on structural stability and functionality. Subsequently, extended molecular dynamics simulations and molecular docking studies were also integrated in order to prioritize the mutations that perturbs structural stability and functionality of h.MTHFR-1. In addition, displacement of Loop (Arg157-Tyr174) and helix α9 (His263-Ser272) involved in open/closed conformation of substrate binding domain were also probed to confirm the functional loss. On juxtaposed analysis, it was inferred that among 126 missense mutations screened, along with known pathogenic mutations (H127â¯T, A222â¯V, T227â¯M, F257â¯V and G387D) predicted that W500C, P254S and D585â¯N variants could be potentially driving homocystinuria. Thus, uncovering the prospects for inclusion of these mutations in diagnostic panels based on further experimental validations.
Assuntos
Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/química , Mutação de Sentido Incorreto , S-Adenosilmetionina/química , Tetra-Hidrofolatos/química , Sítio Alostérico , Motivos de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Expressão Gênica , Homocistinúria/enzimologia , Homocistinúria/patologia , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Aprendizado de Máquina , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , NADP/química , NADP/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , S-Adenosilmetionina/metabolismo , Especificidade por Substrato , Tetra-Hidrofolatos/metabolismo , TermodinâmicaRESUMO
Background Quantification of plasma amino acids is key to the diagnosis of inherited defects of amino acid synthesis, catabolism and transport, many of which present as clinical emergencies. The utility of this test is limited by the long analysis time and subsequent inability of laboratories to provide results in real-time. Traditionally, analysis has been performed by ion exchange chromatography (IEC) but recently there has been a move towards liquid chromatography tandem mass spectrometry (LC-MS/MS) which provides the potential for faster analysis. However, the necessity to derivatise the sample and/or utilise an ion-pair reagent, combined with lack of commercially available stable isotope internal standards (IS) has prevented laboratories fully exploiting the benefits of this methodology. We describe an underivatised LC-MS/MS method enabling patient results to be reported with an improved turnaround time (<1 h). Methods Methanolic IS was added to plasma (10 µL) to precipitate protein. Following centrifugation amino acids were analysed by LC-MS/MS using selected reaction monitoring (SRM) for each analyte and corresponding IS. Results Patient samples (n = 57) and external quality assessment (EQA) material (n = 11) were analysed and results compared with IEC. Comparable accuracy and precision were obtained with 15-min analysis time. Conclusions This method enables the analysis of a clinically comprehensive amino acid profile without the need for derivatisation/ion-pair reagents and benefitting from improved analytical quantitation through multipoint calibration and use of stable isotope IS. The analysis time is fast in comparison to IEC, improves efficiency of laboratory workflow and enables stat analysis of clinically urgent samples.
Assuntos
Aminoácidos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Aminoácidos/isolamento & purificação , Aminoácidos/normas , Precipitação Química , Cromatografia Líquida de Alta Pressão/normas , Cromatografia por Troca Iônica , Homocistinúria/patologia , Humanos , Marcação por Isótopo , Doença da Urina de Xarope de Bordo/patologia , Doença da Deficiência de Ornitina Carbomoiltransferase/patologia , Padrões de Referência , Espectrometria de Massas em Tandem/normas , Estudos de Validação como AssuntoRESUMO
Classic homocystinuria (HCU) is an inherited disorder characterized by elevated homocysteine (Hcy) in plasma and tissues resulting from cystathionine ß-synthase (CBS) deficiency. There is no cure, and patients are predominantly managed by methionine-restricted diet (MRD) to limit the production of Hcy. In this study, we used the I278T mouse model of HCU to evaluate the long-term impact of a novel enzyme replacement therapy [truncated human CBS C15S mutant modified with linear 20-kDa N-hydroxysuccinimide ester polyethylene glycol (OT-58)] on clinical end points relevant to human patients with HCU. In addition, we compared its efficacy on a background of either MRD or normal methionine intake [regular diet (REG)] to that of MRD alone. We found that, compared with untreated I278T mice, OT-58 treatment of I278T mice fed with the REG diet resulted in a 90% decrease in plasma Hcy concentrations and correction of learning/cognition, endothelial dysfunction, hemostasis, bone mineralization, and body composition. On background of the MRD, OT-58 performed equally well with plasma Hcy entirely normalized. The MRD alone decreased plasma Hcy by 67% and corrected the HCU phenotype in I278T mice. However, the MRD increased anxiety and reduced bone mineral content in both I278T mice and wild-type controls. This study shows that OT-58 is a highly efficacious novel treatment for HCU on the background of either normal or restricted methionine intake.-Majtan, T., Park, I., Cox, A., Branchford, B. R., di Paola, J., Bublil, E. M., Kraus, J. P. Behavior, body composition, and vascular phenotype of homocystinuric mice on methionine-restricted diet or enzyme replacement therapy.
Assuntos
Comportamento Animal , Composição Corporal , Cistationina beta-Sintase/uso terapêutico , Terapia de Reposição de Enzimas , Homocistinúria/tratamento farmacológico , Animais , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Modelos Animais de Doenças , Homocistinúria/genética , Homocistinúria/metabolismo , Homocistinúria/patologia , Humanos , Metionina/farmacologia , Camundongos , Camundongos TransgênicosRESUMO
Classical cystathionine ß-synthase-deficient homocystinuria (HCU) is a life-threatening inborn error of sulfur metabolism. Treatment for pyridoxine-nonresponsive HCU involves lowering homocysteine (Hcy) with a methionine (Met)-restricted diet and betaine supplementation. Betaine treatment efficacy diminishes significantly over time due to impairment of betaine-Hcy S-methyltransferase (BHMT) function. Little is known regarding the regulation of BHMT in HCU. Using a betaine-responsive preclinical mouse model of HCU, we observed that this condition induces a 75% repression of BHMT mRNA, protein and enzyme activity, and significant depletion of hepatic betaine levels. BHMT repression was proportional to plasma Hcy levels but was not observed in mouse models of homocystinuria due to either methylenetetrahydrofolate reductase or Met synthase deficiency. Both Met supplementation and chemically induced glutathione depletion exacerbated hepatic BHMT repression in HCU mice but not wild-type (WT) controls. Conversely, cysteine treatment normalized hepatic BHMT expression in HCU mice but had no effect in WT control animals. Taurine treatment induced BHMT expression in HCU mice by 5-fold and restored maximal lowering of Hcy levels during long-term betaine treatment with a concomitant normalization of inflammatory cytokine expression and a significantly improved coagulative phenotype. Collectively, our findings indicate that adjuvantial taurine treatment has the potential to significantly improve clinical outcomes in HCU.-Maclean, K. N., Jiang, H, Phinney, W. N., Keating, A. K., Hurt, K. J., Stabler, S. P. Taurine alleviates repression of betaine-homocysteine S-methyltransferase and significantly improves the efficacy of long-term betaine treatment in a mouse model of cystathionine ß-synthase-deficient homocystinuria.
Assuntos
Betaína-Homocisteína S-Metiltransferase/metabolismo , Betaína/farmacologia , Homocistinúria , Fígado/enzimologia , Taurina/farmacologia , Animais , Betaína-Homocisteína S-Metiltransferase/genética , Modelos Animais de Doenças , Homocistinúria/tratamento farmacológico , Homocistinúria/genética , Homocistinúria/metabolismo , Homocistinúria/patologia , Humanos , Fígado/patologia , Camundongos , Camundongos KnockoutRESUMO
Homocystinuria is a rare inborn error of methionine metabolism caused by cystathionine ß-synthase (CBS) deficiency. The prevalence of homocystinuria in Qatar is 1:1,800 births, mainly due to a founder Qatari missense mutation, c.1006C>T; p.R336C (p.Arg336Cys). We characterized the structure-function relationship of the p.R336C-mutant protein and investigated the effect of different chemical chaperones to restore p.R336C-CBS activity using three models: in silico, ΔCBS yeast, and CRISPR/Cas9 p.R336C knock-in HEK293T and HepG2 cell lines. Protein modeling suggested that the p.R336C induces severe conformational and structural changes, perhaps influencing CBS activity. Wild-type CBS, but not the p.R336C mutant, was able to restore the yeast growth in ΔCBS-deficient yeast in a complementation assay. The p.R336C knock-in HEK293T and HepG2 cells decreased the level of CBS expression and reduced its structural stability; however, treatment of the p.R336C knock-in HEK293T cells with betaine, a chemical chaperone, restored the stability and tetrameric conformation of CBS, but not its activity. Collectively, these results indicate that the p.R336C mutation has a deleterious effect on CBS structure, stability, and activity, and using the chemical chaperones approach for treatment could be ineffective in restoring p.R336C CBS activity.
Assuntos
Cistationina beta-Sintase/genética , Homocistinúria/genética , Chaperonas Moleculares/genética , Proteínas Mutantes/genética , Simulação por Computador , Cistationina beta-Sintase/química , Estabilidade Enzimática , Regulação Enzimológica da Expressão Gênica/genética , Células HEK293 , Células Hep G2 , Homocistinúria/metabolismo , Homocistinúria/patologia , Humanos , Metionina/metabolismo , Chaperonas Moleculares/química , Proteínas Mutantes/química , Mutação de Sentido Incorreto/genética , Dobramento de Proteína , Estrutura Terciária de Proteína , Catar , Relação Estrutura-AtividadeRESUMO
5,10-Methylene-tetrahydrofolate reductase (MTHFR) deficiency is a genetic disorder that can occur at any age and can be easily detected by increased homocysteinemia. In adolescence/adult onset forms, the clinical picture is often complex with association of various neurological features and thrombosis.Here we report the cases of two adult siblings who experienced focal epilepsy at 18 years old as a first disease manifestation, without other symptom during several years. Upon diagnosis, both patients received metabolic treatment comprising B9, B12 and betaine which has stopped the occurrence of seizures, allowing discontinuation of anti-epileptic drugs.Among 24 reviewed adolescent/adult onset patients with MTHFR deficiency in the literature, clinical manifestations included gait disorder (96%, from motor central or peripheral origin), cognitive decline (74%), epileptic syndromes (50%), encephalopathy (30%), psychotic symptoms (17%), and thrombotic events (21%). A total of 41% presented a single neurological manifestation that could stay isolated during at least 3 years, delaying achievement of the diagnosis. Brain MRI showed a mostly periventricular white matter changes in 71% of cases. All patients stabilized or improved following metabolic treatment.Despite being rare, adolescence/adult onset MTHFR deficiency can nevertheless be successfully treated. Therefore, homocysteinemia should be tested in various unexplained neuro-psychiatric syndromes like epilepsy or spastic paraparesis, even if isolated, since waiting for completion of the clinical picture is likely to increase the risk of irreversible neurological damage.
Assuntos
Homocistinúria/diagnóstico , Homocistinúria/patologia , Doenças Metabólicas/diagnóstico , Doenças Metabólicas/patologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/patologia , Adulto , Ataxia/diagnóstico , Ataxia/patologia , Epilepsia/diagnóstico , Epilepsia/patologia , Transtornos Neurológicos da Marcha/diagnóstico , Transtornos Neurológicos da Marcha/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Transtornos Psicóticos/diagnóstico , Transtornos Psicóticos/patologia , Convulsões/diagnóstico , Convulsões/patologiaRESUMO
Cystathionine ß-synthase-deficient homocystinuria (HCU) is a poorly understood, life-threatening inborn error of sulfur metabolism. Analysis of hepatic glutathione (GSH) metabolism in a mouse model of HCU demonstrated significant depletion of cysteine, GSH, and GSH disulfide independent of the block in trans-sulfuration compared with wild-type controls. HCU induced the expression of the catalytic and regulatory subunits of γ-glutamyl ligase, GSH synthase (GS), γ-glutamyl transpeptidase 1, 5-oxoprolinase (OPLAH), and the GSH-dependent methylglyoxal detoxification enzyme, glyoxalase-1. Multiple components of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated antioxidant-response regulatory axis were induced without any detectable activation of Nrf2. Metabolomic analysis revealed the accumulation of multiple γ-glutamyl amino acids and that plasma ophthalmate levels could serve as a noninvasive marker for hepatic redox stress. Neither cysteine, nor betaine treatment was able to reverse the observed enzyme inductions. Taurine treatment normalized the expression levels of γ-glutamyl ligase C/M, GS, OPLAH, and glyoxalase-1, and reversed HCU-induced deficits in protein glutathionylation by acting to double GSH levels relative to controls. Collectively, our data indicate that the perturbation of the γ-glutamyl cycle could contribute to multiple sequelae in HCU and that taurine has significant therapeutic potential for both HCU and other diseases for which GSH depletion is a critical pathogenic factor.-Maclean, K. N., Jiang, H., Aivazidis, S., Kim, E., Shearn, C. T., Harris, P. S., Petersen, D. R., Allen, R. H., Stabler, S. P., Roede, J. R. Taurine treatment prevents derangement of the hepatic γ-glutamyl cycle and methylglyoxal metabolism in a mouse model of classical homocystinuria: regulatory crosstalk between thiol and sulfinic acid metabolism.
Assuntos
Aminobutiratos/metabolismo , Homocistinúria/metabolismo , Fígado/metabolismo , Aldeído Pirúvico/metabolismo , Compostos de Sulfidrila/metabolismo , Ácidos Sulfínicos/metabolismo , Taurina/farmacologia , Aminoácidos/metabolismo , Animais , Cistationina beta-Sintase/metabolismo , Modelos Animais de Doenças , Feminino , Homocistinúria/tratamento farmacológico , Homocistinúria/patologia , Fígado/efeitos dos fármacos , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , gama-Glutamiltransferase/metabolismoRESUMO
Classical homocystinuria (HCU) is an inborn error of sulfur amino acid metabolism caused by deficient activity of cystathionine ß-synthase (CBS), resulting in an accumulation of homocysteine and a concomitant decrease of cystathionine and cysteine in blood and tissues. In mice, the complete lack of CBS is neonatally lethal. In this study, newborn CBS-knockout (KO) mice were treated with recombinant polyethyleneglycolylated human truncated CBS (PEG-CBS). Full survival of the treated KO mice, along with a positive impact on metabolite levels in plasma, liver, brain, and kidneys, was observed. The PEG-CBS treatment prevented an otherwise fatal liver disease characterized by steatosis, death of hepatocytes, and ultrastructural abnormalities of endoplasmic reticulum and mitochondria. Furthermore, treatment of the KO mice for 5 mo maintained the plasma metabolite balance and completely prevented osteoporosis and changes in body composition that characterize both the KO model and human patients. These findings argue that early treatment of patients with HCU with PEG-CBS may prevent clinical symptoms of the disease possibly without the need of dietary protein restriction.-Majtan, T., Hulková, H., Park, I., Krijt, J., Kozich, V., Bublil, E. M., Kraus, J. P. Enzyme replacement prevents neonatal death, liver damage, and osteoporosis in murine homocystinuria.
Assuntos
Cistationina beta-Sintase/metabolismo , Cistationina beta-Sintase/uso terapêutico , Fígado Gorduroso/prevenção & controle , Homocistinúria/tratamento farmacológico , Homocistinúria/enzimologia , Hepatopatias/prevenção & controle , Osteoporose/prevenção & controle , Animais , Composição Corporal/efeitos dos fármacos , Cistationina beta-Sintase/genética , Modelos Animais de Doenças , Fígado Gorduroso/enzimologia , Feminino , Homocistinúria/metabolismo , Homocistinúria/patologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Hepatopatias/enzimologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Recombinantes/uso terapêuticoRESUMO
Homocystinuria is an inborn error of amino acid metabolism caused by deficiency of cystathionine ß-synthase (CBS) activity, biochemically characterized by homocysteine (Hcy) and methionine (Met) accumulation in biological fluids and high urinary excretion of homocystine. Clinical manifestations include thinning and lengthening of long bones, osteoporosis, dislocation of the ocular lens, thromboembolism, and mental retardation. Although the pathophysiology of this disease is poorly known, the present review summarizes the available experimental findings obtained from patients and animal models indicating that oxidative stress may contribute to the pathogenesis of homocystinuria. In this scenario, several studies have shown that enzymatic and non-enzymatic antioxidant defenses are decreased in individuals affected by this disease. Furthermore, markers of lipid, protein, and DNA oxidative damage have been reported to be increased in blood, brain, liver, and skeletal muscle in animal models studied and in homocystinuric patients, probably as a result of increased free radical generation. On the other hand, in vitro and in vivo studies have shown that Hcy induces reactive species formation in brain, so that this major accumulating metabolite may underlie the oxidative damage observed in the animal model and human condition. Taken together, it may be presumed that the disruption of redox homeostasis may contribute to the tissue damage found in homocystinuria. Therefore, it is proposed that the use of appropriate antioxidants may represent a novel adjuvant therapy for patients affected by this disease.
Assuntos
Cistationina beta-Sintase/deficiência , Modelos Animais de Doenças , Homocistinúria/metabolismo , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/metabolismo , Encéfalo/metabolismo , Homocistinúria/patologia , HumanosRESUMO
Combined methylmalonic acidemia (MMA) and homocysteinemia are a group of autosomal recessive disorders caused by inborn errors of cobalamin metabolism, including CblC, D, F, and J, with cblC being the most common subtype. The clinical manifestations of combined MMA and homocysteinemia vary, but typically include neurologic, developmental and hematologic abnormalities.We report 4 children with combined MMA and homocysteinemia who presented predominantly with late-onset diffuse lung diseases (DLD). Of these, 3 accompanied by pulmonary arterial hypertension (PAH), 1 accompanied by hypertension, and 2 accompanied by renal thrombotic microangiopathy (TMA), which was confirmed by renal biopsy. This confirms combined MMA and homocysteinemia should be considered in the differential diagnosis of DLD with or without PAH or renal TMA.