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Peroxisomes are subcellular organelles that play a central role in human physiology by catalyzing a range of unique metabolic functions. The importance of peroxisomes for human health is exemplified by the existence of a group of usually severe diseases caused by an impairment in one or more peroxisomal functions. Among others these include the Zellweger spectrum disorders, X-linked adrenoleukodystrophy, and Refsum disease. To fulfill their role in metabolism, peroxisomes require continued interaction with other subcellular organelles including lipid droplets, lysosomes, the endoplasmic reticulum, and mitochondria. In recent years it has become clear that the metabolic alliance between peroxisomes and other organelles requires the active participation of tethering proteins to bring the organelles physically closer together, thereby achieving efficient transfer of metabolites. This review intends to describe the current state of knowledge about the metabolic role of peroxisomes in humans, with particular emphasis on the metabolic partnership between peroxisomes and other organelles and the consequences of genetic defects in these processes. We also describe the biogenesis of peroxisomes and the consequences of the multiple genetic defects therein. In addition, we discuss the functional role of peroxisomes in different organs and tissues and include relevant information derived from model systems, notably peroxisomal mouse models. Finally, we pay particular attention to a hitherto underrated role of peroxisomes in viral infections.
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Peroxissomos , Animais , Humanos , CamundongosRESUMO
Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293 cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism, and mitochondrial oxidative metabolism.
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Mitocôndrias , Vitamina B 6 , Humanos , Células HEK293 , Proteínas/genética , Proteínas/metabolismo , Fosfato de Piridoxal/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transaminases/metabolismo , Vitamina B 6/metabolismo , Fibroblastos , Células Cultivadas , Piridoxaminafosfato Oxidase/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Oxirredução , Aminoácidos/metabolismoRESUMO
Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss leading to blindness. OAT is a pyridoxal-5'-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of [14C]-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro, which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT, but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care.
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Humans derive fatty acids (FA) from exogenous dietary sources and/or endogenous synthesis from acetyl-CoA, although some FA are solely derived from exogenous sources ("essential FA"). Once inside cells, FA may undergo a wide variety of different modifications, which include their activation to their corresponding CoA ester, the introduction of double bonds, the 2- and ω-hydroxylation and chain elongation, thereby generating a cellular FA pool which can be used for the synthesis of more complex lipids. The biological properties of complex lipids are very much determined by their molecular composition in terms of the FA incorporated into these lipid species. This immediately explains the existence of a range of genetic diseases in man, often with severe clinical consequences caused by variants in one of the many genes coding for enzymes responsible for these FA modifications. It is the purpose of this review to describe the current state of knowledge about FA homeostasis and the genetic diseases involved. This includes the disorders of FA activation, desaturation, 2- and ω-hydroxylation, and chain elongation, but also the disorders of FA breakdown, including disorders of peroxisomal and mitochondrial α- and ß-oxidation.
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Glyoxylate is a key metabolite generated from various precursor substrates in different subcellular compartments including mitochondria, peroxisomes, and the cytosol. The fact that glyoxylate is a good substrate for the ubiquitously expressed enzyme lactate dehydrogenase (LDH) requires the presence of efficient glyoxylate detoxification systems to avoid the formation of oxalate. Furthermore, this detoxification needs to be compartment-specific since LDH is actively present in multiple subcellular compartments including peroxisomes, mitochondria, and the cytosol. Whereas the identity of these protection systems has been established for both peroxisomes and the cytosol as concluded from the deficiency of alanine glyoxylate aminotransferase (AGT) in primary hyperoxaluria type 1 (PH1) and glyoxylate reductase (GR) in PH2, the glyoxylate protection system in mitochondria has remained less well defined. In this manuscript, we show that the enzyme glyoxylate reductase has a bimodal distribution in human embryonic kidney (HEK293), hepatocellular carcinoma (HepG2), and cervical carcinoma (HeLa) cells and more importantly, in human liver, and is actively present in both the mitochondrial and cytosolic compartments. We conclude that the metabolism of glyoxylate in humans requires the complicated interaction between different subcellular compartments within the cell and discuss the implications for the different primary hyperoxalurias.
Assuntos
Oxirredutases do Álcool , Mitocôndrias Hepáticas , Transaminases , Humanos , Mitocôndrias Hepáticas/metabolismo , Células HEK293 , Oxalatos/metabolismo , Fígado/metabolismo , Glioxilatos/metabolismoRESUMO
PURPOSE: Zellweger spectrum disorders (ZSDs) are known as autosomal recessive disorders caused by defective peroxisome biogenesis due to bi-allelic pathogenic variants in any of at least 13 different PEX genes. Here, we report 2 unrelated patients who present with an autosomal dominant ZSD. METHODS: We performed biochemical and genetic studies in blood and skin fibroblasts of the patients and demonstrated the pathogenicity of the identified PEX14 variants by functional cell studies. RESULTS: We identified 2 different single heterozygous de novo variants in the PEX14 genes of 2 patients diagnosed with ZSD. Both variants cause messenger RNA mis-splicing, leading to stable expression of similar C-terminally truncated PEX14 proteins. Functional studies indicated that the truncated PEX14 proteins lost their function in peroxisomal matrix protein import and cause increased degradation of peroxisomes, ie, pexophagy, thus exerting a dominant-negative effect on peroxisome functioning. Inhibition of pexophagy by different autophagy inhibitors or genetic knockdown of the peroxisomal autophagy receptor NBR1 resulted in restoration of peroxisomal functions in the patients' fibroblasts. CONCLUSION: Our finding of an autosomal dominant ZSD expands the genetic repertoire of ZSDs. Our study underscores that single heterozygous variants should not be ignored as possible genetic cause of diseases with an established autosomal recessive mode of inheritance.
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Síndrome de Zellweger , Humanos , Alelos , Peroxissomos/genética , Peroxissomos/metabolismo , Transporte Proteico/fisiologia , Proteínas/genética , Síndrome de Zellweger/genéticaRESUMO
Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.
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Peroxissomos , Proteoma , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Peroxissomos/metabolismo , Proteoma/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Peroxisomes are essential organelles involved in lipid metabolisms including plasmalogen biosynthesis and ß-oxidation of very long-chain fatty acids. Peroxisomes proliferate by the growth and division of pre-existing peroxisomes. The peroxisomal membrane is elongated by Pex11ß and then divided by the dynamin-like GTPase, DLP1 (also known as DRP1 encoded by DNM1L gene), which also functions as a fission factor for mitochondria. Nucleoside diphosphate kinase 3 (NME3) localized in both peroxisomes and mitochondria generates GTP for DLP1 activity. Deficiencies of either of these factors induce abnormal morphology of peroxisomes and/or mitochondria, and are associated with central nervous system dysfunction. To investigate whether the impaired division of peroxisomes affects lipid metabolisms, we assessed the phospholipid composition of cells lacking each of the different division factors. In fibroblasts from the patients deficient in DLP1, NME3, or Pex11ß, docosahexaenoic acid (DHA, C22:6)-containing phospholipids were found to be decreased. Conversely, the levels of several fatty acids such as arachidonic acid (AA, C20:4) and oleic acid (C18:1) were elevated. Mouse embryonic fibroblasts from Drp1- and Pex11ß-knockout mice also showed a decrease in the levels of phospholipids containing DHA and AA. Collectively, these results suggest that the dynamics of organelle morphology exert marked effects on the fatty acid composition of phospholipids.
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Ácidos Docosa-Hexaenoicos , Peroxissomos , Animais , Camundongos , Ácidos Docosa-Hexaenoicos/metabolismo , Dinaminas/metabolismo , Ácidos Graxos/metabolismo , Fibroblastos/metabolismo , Morfogênese , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Peroxissomos/metabolismo , Fosfolipídeos/metabolismoRESUMO
Early-infantile encephalopathies with epilepsy are devastating conditions mandating an accurate diagnosis to guide proper management. Whole-exome sequencing was used to investigate the disease etiology in four children from independent families with intellectual disability and epilepsy, revealing bi-allelic GOT2 mutations. In-depth metabolic studies in individual 1 showed low plasma serine, hypercitrullinemia, hyperlactatemia, and hyperammonemia. The epilepsy was serine and pyridoxine responsive. Functional consequences of observed mutations were tested by measuring enzyme activity and by cell and animal models. Zebrafish and mouse models were used to validate brain developmental and functional defects and to test therapeutic strategies. GOT2 encodes the mitochondrial glutamate oxaloacetate transaminase. GOT2 enzyme activity was deficient in fibroblasts with bi-allelic mutations. GOT2, a member of the malate-aspartate shuttle, plays an essential role in the intracellular NAD(H) redox balance. De novo serine biosynthesis was impaired in fibroblasts with GOT2 mutations and GOT2-knockout HEK293 cells. Correcting the highly oxidized cytosolic NAD-redox state by pyruvate supplementation restored serine biosynthesis in GOT2-deficient cells. Knockdown of got2a in zebrafish resulted in a brain developmental defect associated with seizure-like electroencephalography spikes, which could be rescued by supplying pyridoxine in embryo water. Both pyridoxine and serine synergistically rescued embryonic developmental defects in zebrafish got2a morphants. The two treated individuals reacted favorably to their treatment. Our data provide a mechanistic basis for the biochemical abnormalities in GOT2 deficiency that may also hold for other MAS defects.
Assuntos
Alelos , Ácido Aspártico/metabolismo , Encefalopatias/genética , Proteínas de Ligação a Ácido Graxo/genética , Malatos/metabolismo , Mutação , Animais , Criança , Pré-Escolar , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Sequenciamento do ExomaRESUMO
We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.
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Erros Inatos do Metabolismo dos Aminoácidos/genética , Ataxia/genética , Deficiências do Desenvolvimento/genética , Glutaminase/deficiência , Glutaminase/genética , Glutamina/metabolismo , Repetições de Microssatélites , Mutação , Atrofia/genética , Cerebelo/patologia , Pré-Escolar , Feminino , Genótipo , Glutamina/análise , Humanos , Masculino , Fenótipo , Reação em Cadeia da Polimerase , Sequenciamento Completo do GenomaRESUMO
Barth syndrome is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and neutropenia, caused by deleterious variants in TAFAZZIN. This gene encodes a phospholipid-lysophospholipid transacylase that is required for the remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, individuals with Barth syndrome have a deficiency of mature CL and accumulation of the remodeling intermediate monolysocardiolipin (MLCL). Diagnosis typically relies on mass spectrometric measurement of CL and MLCL in cells or tissues, and we previously described a method in blood spot that uses a specific MLCL/CL ratio as diagnostic biomarker. Here, we describe the evolution of our blood spot assay that is based on the implementation of reversed phase-UHPLC separation followed by full scan high resolution mass spectrometry. In addition to the MLCL/CL ratio, our improved method also generates a complete CL spectrum allowing the interrogation of the CL fatty acid composition, which considerably enhances the diagnostic reliability. This addition negates the need for a confirmatory test in lymphocytes thereby providing a shorter turn-around-time while achieving a more certain test result. As one of the few laboratories that offer this assay, we also evaluated the diagnostic yield and performance from 2006 to 2021 encompassing the use of both the original and improved assay. In this period, we performed 796 diagnostic analyses of which 117 (15%) were characteristic of Barth syndrome. In total, we diagnosed 93 unique individuals with Barth syndrome, including three females, which together amounts to about 40% of all reported individuals with Barth syndrome in the world.
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Síndrome de Barth/diagnóstico , Cardiolipinas/sangue , Linfócitos/metabolismo , Lisofosfolipídeos/sangue , Adolescente , Adulto , Síndrome de Barth/sangue , Criança , Pré-Escolar , Feminino , Humanos , Modelos Lineares , Linfócitos/química , Masculino , Espectrometria de Massas , Reprodutibilidade dos Testes , Adulto JovemRESUMO
Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis.
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Galactosemias , Humanos , Galactosemias/genética , Galactose/metabolismo , Redes e Vias Metabólicas , Biomarcadores/metabolismo , Fosfatos , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , UTP-Hexose-1-Fosfato Uridililtransferase/genética , UTP-Hexose-1-Fosfato Uridililtransferase/metabolismoRESUMO
Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is included in many newborn screening (NBS) programs. Acylcarnitine-based NBS for LCHADD not only identifies LCHADD, but also the other deficiencies of the mitochondrial trifunctional protein (MTP), a multi-enzyme complex involved in long-chain fatty acid ß-oxidation. Besides LCHAD, MTP harbors two additional enzyme activities: long-chain enoyl-CoA hydratase (LCEH) and long-chain ketoacyl-CoA thiolase (LCKAT). Deficiency of one or more MTP activities causes generalized MTP deficiency (MTPD), LCHADD, LCEH deficiency (not yet reported), or LCKAT deficiency (LCKATD). To gain insight in the outcomes of MTP-deficient patients diagnosed after the introduction of NBS for LCHADD in the Netherlands, a retrospective evaluation of genetic, biochemical, and clinical characteristics of MTP-deficient patients, identified since 2007, was carried out. Thirteen patients were identified: seven with LCHADD, five with MTPD, and one with LCKATD. All LCHADD patients (one missed by NBS, clinical diagnosis) and one MTPD patient (clinical diagnosis) were alive. Four MTPD patients and one LCKATD patient developed cardiomyopathy and died within 1 month and 13 months of life, respectively. Surviving patients did not develop symptomatic hypoglycemia, but experienced reversible cardiomyopathy and rhabdomyolysis. Five LCHADD patients developed subclinical neuropathy and/or retinopathy. In conclusion, patient outcomes were highly variable, stressing the need for accurate classification of and discrimination between the MTP deficiencies to improve insight in the yield of NBS for LCHADD. NBS allowed the prevention of symptomatic hypoglycemia, but current treatment options failed to treat cardiomyopathy and prevent long-term complications. Moreover, milder patients, who might benefit from NBS, were missed due to normal acylcarnitine profiles.
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Cardiomiopatias , Hipoglicemia , Erros Inatos do Metabolismo Lipídico , Rabdomiólise , 3-Hidroxiacil-CoA Desidrogenases , Cardiomiopatias/diagnóstico , Cardiomiopatias/genética , Humanos , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Erros Inatos do Metabolismo Lipídico/metabolismo , Miopatias Mitocondriais , Proteína Mitocondrial Trifuncional/deficiência , Biologia Molecular , Triagem Neonatal , Doenças do Sistema Nervoso , Países Baixos , Estudos Retrospectivos , Rabdomiólise/diagnóstico , Rabdomiólise/genéticaRESUMO
A deficiency of 3-hydroxyisobutyric acid dehydrogenase (HIBADH) has been recently identified as a cause of primary 3-hydroxyisobutyric aciduria in two siblings; the only previously recognized primary cause had been a deficiency of methylmalonic semialdehyde dehydrogenase, the enzyme that is immediately downstream of HIBADH in the valine catabolic pathway and is encoded by the ALDH6A1 gene. Here we report on three additional patients from two unrelated families who present with marked and persistent elevations of urine L-3-hydroxyisobutyric acid (L-3HIBA) and a range of clinical findings. Molecular genetic analyses revealed novel, homozygous variants in the HIBADH gene that are private within each family. Evidence for pathogenicity of the identified variants is presented, including enzymatic deficiency of HIBADH in patient fibroblasts. This report describes new variants in HIBADH as an underlying cause of primary 3-hydroxyisobutyric aciduria and expands the clinical spectrum of this recently identified inborn error of valine metabolism. Additionally, we describe a quantitative method for the measurement of D- and L-3HIBA in plasma and urine and present the results of a valine restriction therapy in one of the patients.
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Erros Inatos do Metabolismo dos Aminoácidos , Espectrometria de Massas em Tandem , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Cromatografia Líquida , Humanos , Hidroxibutiratos/urina , Oxirredutases , ValinaRESUMO
BACKGROUND: Primary hyperoxaluria type 1 (PH1) is an inborn error of glyoxylate metabolism, characterized by increased endogenous oxalate production. The metabolic pathways underlying oxalate synthesis have not been fully elucidated, and upcoming therapies require more reliable outcome parameters than the currently used plasma oxalate levels and urinary oxalate excretion rates. We therefore developed a stable isotope infusion protocol to assess endogenous oxalate synthesis rate and the contribution of glycolate to both oxalate and glycine synthesis in vivo . METHODS: Eight healthy volunteers and eight patients with PH1 (stratified by pyridoxine responsiveness) underwent a combined primed continuous infusion of intravenous [1- 13 C]glycolate, [U- 13 C 2 ]oxalate, and, in a subgroup, [D 5 ]glycine. Isotopic enrichment of 13 C-labeled oxalate and glycolate were measured using a new gas chromatography-tandem mass spectrometry (GC-MS/MS) method. Stable isotope dilution and incorporation calculations quantified rates of appearance and synthetic rates, respectively. RESULTS: Total daily oxalate rates of appearance (mean [SD]) were 2.71 (0.54), 1.46 (0.23), and 0.79 (0.15) mmol/d in patients who were pyridoxine unresponsive, patients who were pyridoxine responsive, and controls, respectively ( P =0.002). Mean (SD) contribution of glycolate to oxalate production was 47.3% (12.8) in patients and 1.3% (0.7) in controls. Using the incorporation of [1- 13 C]glycolate tracer in glycine revealed significant conversion of glycolate into glycine in pyridoxine responsive, but not in patients with PH1 who were pyridoxine unresponsive. CONCLUSIONS: This stable isotope infusion protocol could evaluate efficacy of new therapies, investigate pyridoxine responsiveness, and serve as a tool to further explore glyoxylate metabolism in humans.
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Hiperoxalúria Primária , Hiperoxalúria , Humanos , Oxalatos/metabolismo , Espectrometria de Massas em Tandem , Piridoxina , Hiperoxalúria Primária/metabolismo , Glicolatos/urina , Glicina , GlioxilatosRESUMO
PURPOSE: In this study we investigate the disease etiology in 12 patients with de novo variants in FAR1 all resulting in an amino acid change at position 480 (p.Arg480Cys/His/Leu). METHODS: Following next-generation sequencing and clinical phenotyping, functional characterization was performed in patients' fibroblasts using FAR1 enzyme analysis, FAR1 immunoblotting/immunofluorescence, and lipidomics. RESULTS: All patients had spastic paraparesis and bilateral congenital/juvenile cataracts, in most combined with speech and gross motor developmental delay and truncal hypotonia. FAR1 deficiency caused by biallelic variants results in defective ether lipid synthesis and plasmalogen deficiency. In contrast, patients' fibroblasts with the de novo FAR1 variants showed elevated plasmalogen levels. Further functional studies in fibroblasts showed that these variants cause a disruption of the plasmalogen-dependent feedback regulation of FAR1 protein levels leading to uncontrolled ether lipid production. CONCLUSION: Heterozygous de novo variants affecting the Arg480 residue of FAR1 lead to an autosomal dominant disorder with a different disease mechanism than that of recessive FAR1 deficiency and a diametrically opposed biochemical phenotype. Our findings show that for patients with spastic paraparesis and bilateral cataracts, FAR1 should be considered as a candidate gene and added to gene panels for hereditary spastic paraplegia, cerebral palsy, and juvenile cataracts.
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Aldeído Oxirredutases/genética , Éteres , Lipídeos , Paraplegia Espástica Hereditária/genética , Humanos , FenótipoRESUMO
Gyrate atrophy of the choroid and retina (GACR) is a rare inborn error of amino acid metabolism caused by bi-allelic variations in OAT. GACR is characterised by vision decline in early life eventually leading to complete blindness, and high plasma ornithine levels. There is no curative treatment for GACR, although several therapeutic modalities aim to slow progression of the disease by targeting different steps within the ornithine pathway. No international treatment protocol is available. We systematically collected all international literature on therapeutic interventions in GACR to provide an overview of published treatment effects. METHODS: Following the PRISMA guidelines, we conducted a systematic review of the English literature until December 22nd 2020. PubMed and Embase databases were searched for studies related to therapeutic interventions in patients with GACR. RESULTS: A total of 33 studies (n = 107 patients) met the inclusion criteria. Most studies were designed as case reports (n = 27) or case series (n = 4). No randomised controlled trials or large cohort studies were found. Treatments applied were protein-restricted diets, pyridoxine supplementation, creatine or creatine precursor supplementation, l-lysine supplementation, and proline supplementation. Protein-restricted diets lowered ornithine levels ranging from 16.0-91.2%. Pyridoxine responsiveness was reported in 30% of included mutations. Lysine supplementation decreased ornithine levels with 21-34%. Quality assessment showed low to moderate quality of the articles. CONCLUSIONS: Based primarily on case reports ornithine levels can be reduced by using a protein restricted diet, pyridoxine supplementation (variation-dependent) and/or lysine supplementation. The lack of pre-defined clinical outcome measures and structural follow-up in all included studies impeded conclusions on clinical effectiveness. Future research should be aimed at 1) Unravelling the OAT biochemical pathway to identify other possible pathologic metabolites besides ornithine, 2) Pre-defining GACR specific clinical outcome measures, and 3) Establishing an international historical cohort.
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Corioide/efeitos dos fármacos , Atrofia Girata/tratamento farmacológico , Erros Inatos do Metabolismo/tratamento farmacológico , Retina/efeitos dos fármacos , Corioide/patologia , Humanos , Mutação , Retina/patologiaRESUMO
Over the last few years, various inborn disorders have been reported in the malate aspartate shuttle (MAS). The MAS consists of four metabolic enzymes and two transporters, one of them having two isoforms that are expressed in different tissues. Together they form a biochemical pathway that shuttles electrons from the cytosol into mitochondria, as the inner mitochondrial membrane is impermeable to the electron carrier NADH. By shuttling NADH across the mitochondrial membrane in the form of a reduced metabolite (malate), the MAS plays an important role in mitochondrial respiration. In addition, the MAS maintains the cytosolic NAD+ /NADH redox balance, by using redox reactions for the transfer of electrons. This explains why the MAS is also important in sustaining cytosolic redox-dependent metabolic pathways, such as glycolysis and serine biosynthesis. The current review provides insights into the clinical and biochemical characteristics of MAS deficiencies. To date, five out of seven potential MAS deficiencies have been reported. Most of them present with a clinical phenotype of infantile epileptic encephalopathy. Although not specific, biochemical characteristics include high lactate, high glycerol 3-phosphate, a disturbed redox balance, TCA abnormalities, high ammonia, and low serine, which may be helpful in reaching a diagnosis in patients with an infantile epileptic encephalopathy. Current implications for treatment include a ketogenic diet, as well as serine and vitamin B6 supplementation.
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Aspartato Aminotransferases/deficiência , Ácido Aspártico/metabolismo , Malato Desidrogenase/deficiência , Malatos/metabolismo , Erros Inatos do Metabolismo/patologia , Mitocôndrias/patologia , Animais , Aspartato Aminotransferases/genética , Respiração Celular , Humanos , Lactente , Malato Desidrogenase/genética , Erros Inatos do Metabolismo/etiologia , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias/metabolismo , Espasmos Infantis/etiologiaRESUMO
Post-translational protein modifications derived from metabolic intermediates, such as acyl-CoAs, have been shown to regulate mitochondrial function. Patients with a genetic defect in the propionyl-CoA carboxylase (PCC) gene clinically present symptoms related to mitochondrial disorders and are characterised by decreased mitochondrial respiration. Since propionyl-CoA accumulates in PCC deficient patients and protein propionylation can be driven by the level of propionyl-CoA, we hypothesised that protein propionylation could play a role in the pathology of the disease. Indeed, we identified increased protein propionylation due to pathologic propionyl-CoA accumulation in patient-derived fibroblasts and this was accompanied by defective mitochondrial respiration, as was shown by a decrease in complex I-driven respiration. To mimic pathological protein propionylation levels, we exposed cultured fibroblasts, Fao liver cells and C2C12 muscle myotubes to propionate levels that are typically found in these patients. This induced a global increase in protein propionylation and histone protein propionylation and was also accompanied by a decrease in mitochondrial respiration in liver and fibroblasts. However, in C2C12 myotubes propionate exposure did not decrease mitochondrial respiration, possibly due to differences in propionyl-CoA metabolism as compared to the liver. Therefore, protein propionylation could contribute to the pathology in these patients, especially in the liver, and could therefore be an interesting target to pursue in the treatment of this metabolic disease.
Assuntos
Fibroblastos/metabolismo , Metilmalonil-CoA Descarboxilase/genética , Mitocôndrias/genética , Fibras Musculares Esqueléticas/metabolismo , Acidemia Propiônica/genética , Humanos , Fígado/metabolismo , Proteínas de Membrana , Mitocôndrias/enzimologia , Propionatos/metabolismo , Acidemia Propiônica/enzimologia , Processamento de Proteína Pós-Traducional/genéticaRESUMO
D,L-3-hydroxybutyrate (D,L-3-HB, a ketone body) treatment has been described in several inborn errors of metabolism, including multiple acyl-CoA dehydrogenase deficiency (MADD; glutaric aciduria type II). We aimed to improve the understanding of enantiomer-specific pharmacokinetics of D,L-3-HB. Using UPLC-MS/MS, we analyzed D-3-HB and L-3-HB concentrations in blood samples from three MADD patients, and blood and tissue samples from healthy rats, upon D,L-3-HB salt administration (patients: 736-1123 mg/kg/day; rats: 1579-6317 mg/kg/day of salt-free D,L-3-HB). D,L-3-HB administration caused substantially higher L-3-HB concentrations than D-3-HB. In MADD patients, both enantiomers peaked at 30 to 60 minutes, and approached baseline after 3 hours. In rats, D,L-3-HB administration significantly increased Cmax and AUC of D-3-HB in a dose-dependent manner (controls vs ascending dose groups for Cmax : 0.10 vs 0.30-0.35-0.50 mmol/L, and AUC: 14 vs 58-71-106 minutes*mmol/L), whereas for L-3-HB the increases were significant compared to controls, but not dose proportional (Cmax : 0.01 vs 1.88-1.92-1.98 mmol/L, and AUC: 1 vs 380-454-479 minutes*mmol/L). L-3-HB concentrations increased extensively in brain, heart, liver, and muscle, whereas the most profound rise in D-3-HB was observed in heart and liver. Our study provides important knowledge on the absorption and distribution upon oral D,L-3-HB. The enantiomer-specific pharmacokinetics implies differential metabolic fates of D-3-HB and L-3-HB.