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1.
Clin Lab ; 65(7)2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31307170

RESUMO

BACKGROUND: Primary neonatal hypocholinesterase is rare; its genetic pattern and mutation still need to be further studied. METHODS: The patient and his parents are studied using next-generation sequencing technology. RESULTS: A boy one day after birth is admitted to the Neonatal Intensive Care Unit at our hospital after experiencing intermittent vomiting for 12 hours. The patient's serum cholinesterase level (113 - 283 U/L) is lower than normal value (4,000 - 12,600 U/L). Many factors of low serum cholinesterase are excluded. We highly suspect that it may be related to congenital factors. Molecular genetic test results show that the patient carried the BCHE gene (NM_000055.2) and has homozygous frameshift mutations at exon 2 c.401dupA (p.Asn134fs) of chromosome 3q26. It is a pathogenicity mutation. This locus mutation belongs to a novel pathogenic mutation. As a result of this mutation, the 134th amino acid Asn began to frameshift and the translation is terminated early. It can cause the Encoding of protein to truncate and lose its normal function. His parents' serum cholinesterase levels (father: 5,135 U/L; mother: 4,367 U/L) are in the normal value range, but his parents carried a heterozygous BCHE gene. CONCLUSIONS: This study suggests that gene sequence detection should be carried out early in hypocholinesterase of nknown cause in neonates. This study can not only improve understanding of the etiology and pathological mechanism of hypocholinesterase, but also it can enlarge the hypocholinesterase gene mutation spectrum.


Assuntos
Butirilcolinesterase/genética , Mutação da Fase de Leitura , Predisposição Genética para Doença/genética , Erros Inatos do Metabolismo/genética , Butirilcolinesterase/sangue , Butirilcolinesterase/deficiência , Saúde da Família , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Recém-Nascido , Masculino , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/enzimologia
2.
Orphanet J Rare Dis ; 14(1): 84, 2019 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023387

RESUMO

Methylmalonic acidemia/aciduria (MMA) is a genetically heterogeneous group of inherited metabolic disorders biochemically characterized by the accumulation of methylmalonic acid. Isolated MMA is primarily caused by the deficiency of methylmalonyl-CoA mutase (MMA mut; EC 5.4.99.2). A systematic literature review and a meta-analysis were undertaken to assess and compile published epidemiological data on MMA with a focus on the MMA mut subtype (OMIM #251000). Of the 1114 identified records, 227 papers were assessed for eligibility in full text, 48 articles reported on disease epidemiology, and 39 articles were included into the quantitative synthesis. Implementation of newborn screening in various countries has allowed for the estimation of birth prevalence of MMA and its isolated form. Meta-analysis pooled point estimates of MMA (all types) detection rates were 0.79, 1.12, 1.22 and 6.04 per 100,000 newborns in Asia-Pacific, Europe, North America and the Middle East and North Africa (MENA) regions, respectively. The detection rate of isolated MMA was < 1 per 100,000 newborns in all regions with the exception of MENA where it approached 6 per 100,000 newborns. Few studies published data on the epidemiology of MMA mut, therefore no meta-analysis could have been performed on this subtype. Most of the identified papers reported birth prevalence estimates below 1 per 100,000 newborns for MMA mut. The systematic literature review clearly demonstrates that MMA and its subtypes are ultra-rare disorders.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Erros Inatos do Metabolismo dos Aminoácidos/epidemiologia , Metilmalonil-CoA Mutase/genética , Erros Inatos do Metabolismo dos Aminoácidos/genética , Feminino , Humanos , Recém-Nascido , Masculino , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/epidemiologia , Erros Inatos do Metabolismo/genética , Metilmalonil-CoA Mutase/deficiência , Triagem Neonatal
3.
PLoS Genet ; 15(3): e1007605, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30856165

RESUMO

Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.


Assuntos
Cardiomiopatia Dilatada/enzimologia , Cardiomiopatia Dilatada/genética , Catarata/enzimologia , Catarata/genética , Hipogonadismo/enzimologia , Hipogonadismo/genética , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Pirofosfatases/deficiência , Animais , Sequência de Bases , Pré-Escolar , Análise Mutacional de DNA , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Feminino , Homozigoto , Humanos , Inosina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/enzimologia , Mutação , Linhagem , Pirofosfatases/genética , RNA/genética , RNA/metabolismo , Sequenciamento Completo do Exoma
4.
Am J Hum Genet ; 104(2): 287-298, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30661771

RESUMO

Hypusine is formed post-translationally from lysine and is found in a single cellular protein, eukaryotic translation initiation factor-5A (eIF5A), and its homolog eIF5A2. Biosynthesis of hypusine is a two-step reaction involving the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). eIF5A is highly conserved throughout eukaryotic evolution and plays a role in mRNA translation, cellular proliferation, cellular differentiation, and inflammation. DHPS is also highly conserved and is essential for life, as Dhps-null mice are embryonic lethal. Using exome sequencing, we identified rare biallelic, recurrent, predicted likely pathogenic variants in DHPS segregating with disease in five affected individuals from four unrelated families. These individuals have similar neurodevelopmental features that include global developmental delay and seizures. Two of four affected females have short stature. All five affected individuals share a recurrent missense variant (c.518A>G [p.Asn173Ser]) in trans with a likely gene disrupting variant (c.1014+1G>A, c.912_917delTTACAT [p.Tyr305_Ile306del], or c.1A>G [p.Met1?]). cDNA studies demonstrated that the c.1014+1G>A variant causes aberrant splicing. Recombinant DHPS enzyme harboring either the p.Asn173Ser or p.Tyr305_Ile306del variant showed reduced (20%) or absent in vitro activity, respectively. We co-transfected constructs overexpressing HA-tagged DHPS (wild-type or mutant) and GFP-tagged eIF5A into HEK293T cells to determine the effect of these variants on hypusine biosynthesis and observed that the p.Tyr305_Ile306del and p.Asn173Ser variants resulted in reduced hypusination of eIF5A compared to wild-type DHPS enzyme. Our data suggest that rare biallelic variants in DHPS result in reduced enzyme activity that limits the hypusination of eIF5A and are associated with a neurodevelopmental disorder.


Assuntos
Genes Recessivos/genética , Lisina/análogos & derivados , Mutação , Transtornos do Neurodesenvolvimento/enzimologia , Transtornos do Neurodesenvolvimento/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Alelos , Sequência de Aminoácidos , Criança , Pré-Escolar , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/genética , Feminino , Haplótipos , Humanos , Lisina/biossíntese , Masculino , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Linhagem , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Convulsões/enzimologia , Convulsões/genética , Adulto Jovem
5.
Xenobiotica ; 49(10): 1244-1250, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30351217

RESUMO

1. Flavin-containing monooxygenase 3 (FMO3) in humans is polymorphic in several ethnic groups, including Caucasians, Africans and Asians. Some FMO3 variants are associated with a disorder trimethylaminuria. 2. In the current study, we used the results from urinary phenotyping assays to identify 63 subjects with <85% FMO3 metabolic capacity with respect to trimethylamine N-oxidation among 787 Japanese volunteers with self-reported trimethylaminuria. The 63 subjects with reduced FMO3 activity were screened and investigated in detail to identify novel FMO3 variants. 3. Homozygous or heterozygous individuals for new single nucleotide substitution variants/haplotypes p.(Pro282Leu), p.[(Glu158Lys; Glu308Gly; Thr329Ala)], p.[(Glu158Lys; Glu308Gly; Asp429Gly)], p.[(Val257Met; Leu473Pro)], p.[(Glu158Lys; Glu308Gly; Ile441Thr)], and p.[(Arg205Cys; Gly503Arg)] were identified in six proband subjects and their family members after pedigree analyses. 4. These variant FMO3 proteins recombinantly expressed in Escherichia coli membranes exhibited decreased N-oxygenation activities toward trimethylamine (Vmax/Km < 40% that of the wild-type). 5. Although the allele frequencies of the six new variants and/or haplotypes were low, the present results indicated that individuals homozygous or heterozygous for any of these novel missense FMO3 variants or known nonsense mutations such as p.(Cys197Ter) or p.(Arg205Cys) highly found in this self-reported Japanese trimethylaminuria cohort may have reduced FMO3 activity with respect to the N-oxygenation of trimethylamine.


Assuntos
Alelos , Códon sem Sentido , Frequência do Gene , Haplótipos , Erros Inatos do Metabolismo/genética , Metilaminas/urina , Mutação de Sentido Incorreto , Adolescente , Adulto , Idoso , Substituição de Aminoácidos , Grupo com Ancestrais do Continente Asiático , Pré-Escolar , Feminino , Humanos , Lactente , Japão , Masculino , Erros Inatos do Metabolismo/enzimologia , Metilaminas/metabolismo , Pessoa de Meia-Idade , Oxigenases
6.
J Hum Genet ; 64(2): 87-98, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30514913

RESUMO

Carnitine palmitoyltransferase (CPT) II deficiency is one of the most common forms of mitochondrial fatty acid oxidation disorder. Its clinical phenotypes are classified into the muscle, severe infantile, and lethal neonatal forms. Among Caucasians, the muscle form predominates, and the c.338C > T (p.S113L) variant is detected in most cases, whereas among the Japanese, c.1148T > A (p.F383Y) is the variant allele occurring with the highest frequency and can apparently cause symptoms of the severe infantile form. Newborn screening (NBS) for this potentially fatal disease has not been established. We encountered an infantile case of CPT II deficiency not detected in NBS using C16 and C18:1 concentrations as indices, and therefore we adopted the (C16 + C18:1)/C2 ratio as an alternative primary index. As a result, the disease was diagnosed in nine of 31 NBS-positive subjects. The values for (C16 + C18:1)/C2 in the affected newborns partly overlapped with those in unaffected ones. Among several other indices proposed previously, C14/C3 has emerged as a more promising index. Based on these findings, nationwide NBS for CPT II deficiency using both (C16 + C18:1)/C2 and C14/C3 as indices was officially approved and started in April 2018. We diagnosed the disease in four young children presenting with symptoms of the muscle form, whose values for the new indices were not elevated. Although it is still difficult to detect all cases of the muscle form of CPT II deficiency in NBS, our system is expected to save many affected children in Japan with the severe infantile form predominating.


Assuntos
Carnitina O-Palmitoiltransferase/deficiência , Erros Inatos do Metabolismo/diagnóstico , Triagem Neonatal/métodos , Humanos , Recém-Nascido , Erros Inatos do Metabolismo/enzimologia , Prognóstico
7.
Neurology ; 91(11): e1077-e1082, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30111548

RESUMO

OBJECTIVE: To study the variable clinical picture and exercise tolerance of patients with phosphoglycerate kinase (PGK) 1 deficiency and how it relates to residual PGK enzyme activity. METHODS: In this case series study, we evaluated 7 boys and men from 5 families with PGK1 deficiency. Five had pure muscle symptoms, while 2 also had mild intellectual disability with or without anemia. Muscle glycolytic and oxidative capacities were evaluated by an ischemic forearm exercise test and by cycle ergometry. RESULTS: Enzyme levels of PGK were 4% to 9% of normal in red cells and 5% to10% in muscle in pure myopathy patients and 2.6% in both muscle and red cells in the 2 patients with multisystem involvement. Patients with pure myopathy had greater increases in lactate with ischemic exercise (2-3 mmol/L) vs the 2 multisystem-affected patients (<1 mmol/L). Myopathy patients had higher oxidative capacity in cycle exercise vs multisystem affected patients (≈30 vs ≈15 mL/kg per minute). One multisystem-affected patient developed frank myoglobinuria after the short exercise test. CONCLUSIONS: This case series study of PGK1 deficiency suggests that the level of impaired glycolysis in PGK deficiency is a major determinant of phenotype. Lower glycolytic capacity in PGK1 deficiency seems to result in multisystem involvement and increased susceptibility to exertional rhabdomyolysis.


Assuntos
Tolerância ao Exercício/fisiologia , Doenças Genéticas Ligadas ao Cromossomo X/enzimologia , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/fisiopatologia , Fosfoglicerato Quinase/deficiência , Fosfoglicerato Quinase/metabolismo , Ergometria , Teste de Esforço , Doenças Genéticas Ligadas ao Cromossomo X/complicações , Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Humanos , Deficiência Intelectual/sangue , Deficiência Intelectual/complicações , Deficiência Intelectual/enzimologia , Deficiência Intelectual/fisiopatologia , Ácido Láctico/sangue , Masculino , Erros Inatos do Metabolismo/complicações , Erros Inatos do Metabolismo/diagnóstico , Músculo Esquelético/metabolismo , Doenças Musculares/sangue , Doenças Musculares/complicações , Doenças Musculares/enzimologia , Doenças Musculares/fisiopatologia , Fenótipo , Fosfoglicerato Quinase/sangue
8.
J Biol Chem ; 293(40): 15715-15724, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30135208

RESUMO

The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or Staphylococcus aureus by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to >30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in ∼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.


Assuntos
Corantes Fluorescentes/química , Ácido Hipocloroso/análise , Neutrófilos/enzimologia , Fagocitose , Fagossomos/metabolismo , Rodaminas/química , Bioensaio , Corantes Fluorescentes/metabolismo , Humanos , Ácido Hipocloroso/imunologia , Ácido Hipocloroso/metabolismo , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/imunologia , Erros Inatos do Metabolismo/patologia , NADPH Oxidases/genética , NADPH Oxidases/imunologia , NADPH Oxidases/metabolismo , Neutrófilos/imunologia , Neutrófilos/patologia , Proteínas Opsonizantes/química , Peroxidase/deficiência , Peroxidase/genética , Peroxidase/imunologia , Fagossomos/imunologia , Fagossomos/ultraestrutura , Cultura Primária de Células , Rodaminas/metabolismo , Espectrometria de Fluorescência , Staphylococcus aureus/imunologia , Zimosan/química
9.
Int J Mol Sci ; 19(4)2018 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-29597274

RESUMO

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency (mitochondrial HMG-CoA synthase deficiency or mHS deficiency, OMIM #605911) is an inborn error of metabolism that affects ketone body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycemia and dicarboxylic aciduria. The diagnosis is difficult due to the relatively unspecific clinical and biochemical presentation, and fewer than 30 patients have been described. This work describes three new patients with mHS deficiency and two missense mutations c.334C>T (p.R112W) and c.430G>T (p.V144L) previously not reported. We developed a new method to express and measure the activity of the enzyme and in this work the study is extended to ten new missense variants including those of our patients. Enzymatic assays showed that three of the mutant proteins retained some but seven completely lacked activity. The identification of a patient homozygous for a mutation that retains 70% of enzyme activity opens the door to a new interpretation of the disease by demonstrating that a modest impairment of enzyme function can actually produce symptoms. This is also the first study employing molecular dynamics modelling of the enzyme mutations. We show that the correct maintenance of the dimerization surface is crucial for retaining the structure of the active center and therefore the activity of the enzyme.


Assuntos
Hidroximetilglutaril-CoA Sintase/deficiência , Erros Inatos do Metabolismo , Proteínas Mitocondriais/deficiência , Mutação de Sentido Incorreto , Multimerização Proteica , Substituição de Aminoácidos , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/patologia
10.
J Inherit Metab Dis ; 41(1): 59-72, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28726069

RESUMO

Several inherited metabolic disorders are associated with an accumulation of reactive acyl-CoA metabolites that can non-enzymatically react with lysine residues to modify proteins. While the role of acetylation is well-studied, the pathophysiological relevance of more recently discovered acyl modifications, including those found in inherited metabolic disorders, warrants further investigation. We recently showed that sirtuin 4 (SIRT4) removes glutaryl, 3-hydroxy-3-methylglutaryl, 3-methylglutaryl, and 3-methylglutaconyl modifications from lysine residues. Thus, we used SIRT4 knockout mice, which can accumulate these novel post-translational modifications, as a model to investigate their physiological relevance. Since SIRT4 is localized to mitochondria and previous reports have shown SIRT4 influences metabolism, we thoroughly characterized glucose and lipid metabolism in male and female SIRT4KO mice across different genetic backgrounds. While only minor perturbations in overall lipid metabolism were observed, we found SIRT4KO mice consistently had elevated glucose- and leucine-stimulated insulin levels in vivo and developed accelerated age-induced insulin resistance. Importantly, elevated leucine-stimulated insulin levels in SIRT4KO mice were dependent upon genetic background since SIRT4KO mice on a C57BL/6NJ genetic background had elevated leucine-stimulated insulin levels but not SIRT4KO mice on the C57BL/6J background. Taken together, the data suggest that accumulation of acyl modifications on proteins in inherited metabolic disorders may contribute to the overall metabolic dysfunction seen in these patients.


Assuntos
Glicemia/metabolismo , Resistência à Insulina , Leucina/sangue , Metabolismo dos Lipídeos , Erros Inatos do Metabolismo/enzimologia , Proteínas Mitocondriais/deficiência , Sirtuínas/deficiência , Fatores Etários , Animais , Biomarcadores/sangue , Feminino , Predisposição Genética para Doença , Técnicas In Vitro , Insulina/sangue , Resistência à Insulina/genética , Leucina/administração & dosagem , Metabolismo dos Lipídeos/genética , Lisina , Masculino , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/fisiopatologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Fenótipo , Processamento de Proteína Pós-Traducional , Sirtuínas/genética , Regulação para Cima
11.
Clin Exp Rheumatol ; 35(5): 735-738, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28850023

RESUMO

OBJECTIVES: Pentraxin 3 (PTX3) is a multifunctional soluble factor. PTX3 can be involved in the regulation of vasculitis and is expressed in the cytoplasm of neutrophils. As anti-neutrophil cytoplasmic antibody (ANCA) is recognised as a cause of vasculitis, we aimed to discover the role of PTX3 in ANCA production in vivo. METHODS: To this end, we used aluminum salt (alum), which induces neutrophil extracellular traps, as an adjuvant for producing anti-myeloperoxidase-ANCA (MPO-ANCA). Specifically, we intraperitoneally injected alum and recombinant MPO (rMPO) into MPO-deficient mice and then measured the concentration of anti-MPO IgG in their blood. To show the involvement of extracellular PTX3 in this model, we assessed PTX3 protein content and host double-stranded DNA levels in the mice's peritoneal fluid after alum injection. In addition, we simultaneously administered recombinant PTX3, rMPO and alum to MPO-deficient mice to assess the function of PTX3 in producing anti-MPO IgG in vivo. RESULTS: Anti-MPO IgG was produced by the alum + rMPO immunisation model in MPO-deficient but not wildtype mice. Injection of alum induced extracellular PTX3 as well as double-stranded DNA and dead cells in MPO-deficient mice. Simultaneous injection of recombinant PTX3 with rMPO and alum attenuated the production of anti-MPO IgG in MPO-deficient mice. CONCLUSIONS: Our current findings provide evidence that PTX3 attenuates the production of murine MPO-ANCA.


Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/farmacologia , Anticorpos Anticitoplasma de Neutrófilos/sangue , Proteína C-Reativa/imunologia , Imunoglobulina G/sangue , Erros Inatos do Metabolismo/imunologia , Proteínas do Tecido Nervoso/imunologia , Peroxidase/imunologia , Animais , Líquido Ascítico/imunologia , Líquido Ascítico/metabolismo , Proteína C-Reativa/administração & dosagem , Proteína C-Reativa/metabolismo , DNA/imunologia , DNA/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Feminino , Masculino , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/administração & dosagem , Proteínas do Tecido Nervoso/metabolismo , Peroxidase/administração & dosagem , Peroxidase/deficiência , Peroxidase/genética
12.
Ann Neurol ; 81(5): 641-652, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28318037

RESUMO

OBJECTIVE: Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2-/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. METHODS: To test these hypotheses, we assessed two therapies in Tk2-/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. RESULTS: We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2-/- animals compared to dCMP+dTMP. INTERPRETATION: Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.


Assuntos
Antimetabólitos/farmacologia , Desoxicitidina Monofosfato/farmacologia , Erros Inatos do Metabolismo/tratamento farmacológico , Doenças Mitocondriais/tratamento farmacológico , Tetra-Hidrouridina/farmacologia , Timidina Quinase/deficiência , Timidina/farmacologia , Animais , Antimetabólitos/administração & dosagem , DNA Mitocondrial/efeitos dos fármacos , Desoxicitidina Monofosfato/administração & dosagem , Modelos Animais de Doenças , Quimioterapia Combinada , Erros Inatos do Metabolismo/enzimologia , Camundongos , Camundongos Transgênicos , Doenças Mitocondriais/enzimologia , Tetra-Hidrouridina/administração & dosagem , Timidina/administração & dosagem
14.
Clin Biochem ; 50(3): 121-126, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27784639

RESUMO

OBJECTIVE: The present study highlights the feasibility of gas chromatography/mass spectrometry (GC/MS)-based analysis for simultaneous detection of >200 marker metabolites in urine found in characteristic pattern in inborn errors of metabolism (IEM) in India. DESIGN AND METHODS: During this retrospective study conducted from July 2013 to January 2016, we collected urine specimens on filter papers from Indian children across the country along with relevant demographic and clinical data. The laboratory technique involved urease pretreatment followed by deproteinization, derivatization, and subsequent computer-aided analysis of organic acids, amino acids, fatty acids, and sugars by GC/MS, which enable chemical diagnosis of IEM. RESULTS: Totally 23,140 patients were investigated for IEM with an estimated frequency of about 1.40%, that is, 323 positive cases. Most frequent disorders observed were of primary lactic acidemia (27.2%) and organic acidemia (methylmalonic aciduria, glutaric acidemia type I, propionic aciduria, etc.) followed by aminoacidopathies (maple syrup urine disease, phenylketonuria, tyrosinemia, etc.). Furthermore, alkaptonuria, canavan disease, and 4-hydroxybutyric aciduria were also diagnosed. Prompt treatment following diagnosis led to a better outcome in a considerable number of patients. CONCLUSIONS: GC/MS with one-step metabolomics enables quick detection, accurate identification, and precise quantification of a wide range of urinary markers that may not be discovered using existing newborn screening programs. The technique is effective as a second-tier test to other established screening technologies, as well as one-step primary screening tool for a wide spectrum of IEM.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Erros Inatos do Metabolismo/metabolismo , Algoritmos , Aminoácidos/análise , Criança , Feminino , Humanos , Índia , Lactente , Recém-Nascido , Masculino , Erros Inatos do Metabolismo/enzimologia , Estudos Retrospectivos , Succinato-Semialdeído Desidrogenase/metabolismo
15.
Gene ; 594(2): 203-210, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27601257

RESUMO

The deficiency of 3-methycrotonyl-CoA carboxylase (3-MCC; EC 6.4.1.4) is an autosomal recessive organic aciduria that is included in the newborn screening programs of several countries. This study reports data mainly obtained from the Portuguese newborn screening program collected over a ten-year period. Analysis of the MCCC1 and MCCC2 genes yielded 26 previously unreported mutations and a variant of clinically unknown significance. These mutations are discussed in the context of their likely impact on the function of the 3-MCC enzyme, with a view to exploring whether a phenotype-genotype correlation might be discerned. Further, these mutations were analysed in the context of what is known of the MCCC1 and MCCC2 mutational spectra, information that will be useful in both clinical and laboratory practice.


Assuntos
Carbono-Carbono Ligases/deficiência , Erros Inatos do Metabolismo/genética , Mutação , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Programas de Rastreamento , Erros Inatos do Metabolismo/enzimologia , Portugal
16.
Ann Transplant ; 21: 525-30, 2016 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-27550409

RESUMO

Hepatic-based metabolic disorders are characterized by an enzyme deficiency expressed solely or mainly in the liver. They are divided into cirrhotic or non-cirrhotic metabolic liver diseases (NCMLDs), and most of them can be treated by liver transplantation. Because the livers with NCMLDs are usually structurally and functionally normal, the primary aim of the liver graft is to support the deficient enzymes rather than maintaining liver functions. Hence, we hypothesize that the exchange of partial liver grafts by the technique of auxiliary partial orthotopic liver transplantation (APOLT) between patients with 2 different NCMLDs may be feasible to replace the deficient enzymes in each patient. This hypothesis is based on the following conditions: (i) the patients have no chance of undergoing timely liver transplantation, (ii) the symptoms of each NCMLD may be alleviated after exchanging partial liver grafts, and (iii) each graft is anatomically appropriate for APOLT. In addition, we evaluate it with a focus on selection of cases, designing of graft sizes, and surgical techniques for reciprocal APOLT.


Assuntos
Hepatopatias/cirurgia , Transplante de Fígado/métodos , Erros Inatos do Metabolismo/cirurgia , Criança , Enzimas/deficiência , Humanos , Fígado/enzimologia , Fígado/patologia , Fígado/cirurgia , Hepatopatias/classificação , Hepatopatias/enzimologia , Erros Inatos do Metabolismo/classificação , Erros Inatos do Metabolismo/enzimologia , Tamanho do Órgão , Seleção de Pacientes
17.
Biochimie ; 126: 27-30, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26853819

RESUMO

Disruptions in folate-mediated one-carbon metabolism (FOCM) are associated with risk for several pathologies including developmental anomalies such as neural tube defects and congenital heart defects, diseases of aging including cognitive decline, neurodegeneration and epithelial cancers, and hematopoietic disorders including megaloblastic anemia. However, the causal pathways and mechanisms that underlie these pathologies remain unresolved. Because folate-dependent anabolic pathways are tightly interconnected and best described as a metabolic network, the identification of causal pathways and associated mechanisms of pathophysiology remains a major challenge in identifying the contribution of individual pathways to disease phenotypes. Investigations of genetic mouse models and human inborn errors of metabolism enable a more precise dissection of the pathways that constitute the FOCM network and enable elucidation of causal pathways associated with NTDs. In this overview, we summarize recent evidence that the enzyme MTHFD1 plays an essential role in FOCM in humans and in mice, and that it determines the partitioning of folate-activated one carbon units between the folate-dependent de novo thymidylate and homocysteine remethylation pathways through its regulated nuclear localization. We demonstrate that impairments in MTHFD1 activity compromise both homocysteine remethylation and de novo thymidylate biosynthesis, and provide evidence that MTHFD1-associated disruptions in de novo thymidylate biosynthesis lead to genome instability that may underlie folate-associated immunodeficiency and birth defects.


Assuntos
Instabilidade Genômica , Metilenotetra-Hidrofolato Desidrogenase (NADP)/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Timidina Monofosfato/biossíntese , Animais , Anormalidades Congênitas/enzimologia , Anormalidades Congênitas/genética , Ácido Fólico/biossíntese , Ácido Fólico/genética , Homocisteína/biossíntese , Homocisteína/genética , Humanos , Síndromes de Imunodeficiência/enzimologia , Síndromes de Imunodeficiência/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Metilenotetra-Hidrofolato Desidrogenase (NADP)/genética , Camundongos , Antígenos de Histocompatibilidade Menor/genética
18.
Cell Rep ; 14(6): 1308-1316, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26854223

RESUMO

Ambient temperature affects energy intake and expenditure to maintain homeostasis in a continuously fluctuating environment. Here, mice with an adipose-specific defect in fatty acid oxidation (Cpt2(A-/-)) were subjected to varying temperatures to determine the role of adipose bioenergetics in environmental adaptation and body weight regulation. Microarray analysis of mice acclimatized to thermoneutrality revealed that Cpt2(A-/-) interscapular brown adipose tissue (BAT) failed to induce the expression of thermogenic genes such as Ucp1 and Pgc1α in response to adrenergic stimulation, and increasing ambient temperature exacerbated these defects. Furthermore, thermoneutral housing induced mtDNA stress in Cpt2(A-/-) BAT and ultimately resulted in a loss of interscapular BAT. Although the loss of adipose fatty acid oxidation resulted in clear molecular, cellular, and physiologic deficits in BAT, body weight gain and glucose tolerance were similar in control and Cpt2(A-/-) mice in response to a high-fat diet, even when mice were housed at thermoneutrality.


Assuntos
Tecido Adiposo Marrom/enzimologia , Carnitina O-Palmitoiltransferase/deficiência , Carnitina O-Palmitoiltransferase/genética , Ácidos Graxos/metabolismo , Erros Inatos do Metabolismo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Termogênese/genética , Proteína Desacopladora 1/genética , Acil-CoA Desidrogenase/genética , Acil-CoA Desidrogenase/metabolismo , Adaptação Fisiológica , Tecido Adiposo Marrom/patologia , Animais , Peso Corporal , Carnitina O-Palmitoiltransferase/metabolismo , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Dieta Hiperlipídica/efeitos adversos , Regulação da Expressão Gênica , Metabolismo dos Lipídeos/genética , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/patologia , Camundongos , Camundongos Knockout , Obesidade/enzimologia , Obesidade/etiologia , Obesidade/genética , Obesidade/patologia , Oxirredução , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Transdução de Sinais , Temperatura , Proteína Desacopladora 1/metabolismo
19.
Int J Mol Sci ; 17(1)2016 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-26771602

RESUMO

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.


Assuntos
Processamento Alternativo , Amidoidrolases/química , Erros Inatos do Metabolismo/genética , Mutação , Precursores de RNA/genética , Amidoidrolases/genética , Amidoidrolases/metabolismo , Sequência de Bases , Domínio Catalítico , Criança , Éxons , Feminino , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Heterozigoto , Humanos , Lactente , Íntrons , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/patologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Precursores de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência
20.
Hematology ; 21(3): 193-7, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25978498

RESUMO

IMPORTANCE: Functional methionine synthase reductase deficiency, also known as cobalamin E disorder, is a rare autosomal recessive inherited disease that results in an impaired remethylation of homocysteine to methionine. It presents with macrocytic anemia, hyperhomocysteinemia, and hypomethioninemia, and may also be accompanied with neurological impairment. CLINICAL PRESENTATION: We describe two new cases of unrelated girls with megaloblastic anemia misclassified at first as congenital dyserythropoietic anemia with development of neurologic dysfunction in one of them. INTERVENTION: The posterior finding of biochemical features (hyperhomocysteinemia and hypomethioninemia) focused the diagnosis on the inborn errors of intracellular vitamin B12. Subsequent molecular analysis of the methionine synthase reductase (MTRR) gene revealed compound heterozygosity for a transition c.1361C > T (p.Ser454Leu) and another, not yet described in literature, c.1677-1G > A (p.Glu560fs) in one patient, and a single homozygosis mutation, c.1361C > T (p.Ser545Leu) in the other one. These mutations confirmed the diagnosis of cobalamin E deficiency. CONCLUSION: Treatment with hydroxocobalamin in combination with betaine appears to be useful for hematological improvement and prevention of brain disabilities in CblE-affected patients. Our study widens the clinical, molecular, metabolic, and cytological knowledge of deficiency MTRR enzyme.


Assuntos
Substituição de Aminoácidos , Anemia Macrocítica , Betaína/administração & dosagem , Ferredoxina-NADP Redutase , Hidroxocobalamina/administração & dosagem , Erros Inatos do Metabolismo , Adulto , Anemia Macrocítica/tratamento farmacológico , Anemia Macrocítica/enzimologia , Anemia Macrocítica/genética , Criança , Feminino , Ferredoxina-NADP Redutase/deficiência , Ferredoxina-NADP Redutase/genética , Humanos , Hiper-Homocisteinemia/tratamento farmacológico , Hiper-Homocisteinemia/enzimologia , Hiper-Homocisteinemia/genética , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Mutação de Sentido Incorreto
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