RESUMO
Aldehyde dehydrogenase 5a1-deficient (aldh5a1-/-) mice, the murine orthologue of human succinic semialdehyde dehydrogenase deficiency (SSADHD), manifest increased GABA (4-aminobutyric acid) that disrupts autophagy, increases mitochondria number, and induces oxidative stress, all mitigated with the mTOR (mechanistic target of rapamycin) inhibitor rapamycin [1]. Because GABA regulates mTOR, we tested the hypothesis that aldh5a1-/- mice would show altered levels of mRNA for genes associated with mTOR signaling and oxidative stress that could be mitigated by inhibiting mTOR. We observed that multiple metabolites associated with GABA metabolism (γ-hydroxybutyrate, succinic semialdehyde, D-2-hydroxyglutarate, 4,5-dihydrohexanoate) and oxidative stress were significantly increased in multiple tissues derived from aldh5a1-/- mice. These metabolic perturbations were associated with decreased levels of reduced glutathione (GSH) in brain and liver of aldh5a1-/- mice, as well as increased levels of adducts of the lipid peroxidation by-product, 4-hydroxy-2-nonenal (4-HNE). Decreased liver mRNA levels for multiple genes associated with mTOR signaling and oxidative stress parameters were detected in aldh5a1-/- mice, and several were significantly improved with the administration of mTOR inhibitors (Torin 1/Torin 2). Western blot analysis of selected proteins corresponding to oxidative stress transcripts (glutathione transferase, superoxide dismutase, peroxiredoxin 1) confirmed gene expression findings. Our data provide additional preclinical evidence for the potential therapeutic efficacy of mTOR inhibitors in SSADHD.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/tratamento farmacológico , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Deficiências do Desenvolvimento/tratamento farmacológico , Deficiências do Desenvolvimento/metabolismo , Deleção de Genes , Succinato-Semialdeído Desidrogenase/deficiência , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Ácido gama-Aminobutírico/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Deficiências do Desenvolvimento/genética , Modelos Animais de Doenças , Feminino , Glutationa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Succinato-Semialdeído Desidrogenase/genética , Succinato-Semialdeído Desidrogenase/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
We hypothesized that blood levels of γ-aminobutyric acid (GABA) and γ-hydroxybutyric acid (GHB), biomarkers of succinic semialdehyde dehydrogenase deficiency (SSADHD), would correlate with age. GABA and GHB were quantified in plasma and red blood cells (RBCs) from 18 patients (age range 5-41 years; median 8). Both metabolites negatively correlated with age (P < 0.05). Plasma and RBC GHB declined with age, reaching a nadir and approximate steady state by 10 years. Declining plasma GABA achieved this approximate steady state at 30-40 years of age. These biomarker relationships may reflect further GABA- and GHB-ergic neurotransmission imbalances that correlate with the onset of adolescent/adulthood neuropsychiatric morbidity and epilepsy in SSADHD.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/sangue , Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Biomarcadores/sangue , Deficiências do Desenvolvimento/sangue , Deficiências do Desenvolvimento/metabolismo , Succinato-Semialdeído Desidrogenase/deficiência , Ácido gama-Aminobutírico/metabolismo , Adolescente , Adulto , Criança , Pré-Escolar , Epilepsia/sangue , Epilepsia/metabolismo , Feminino , Humanos , Hidroxibutiratos/metabolismo , Masculino , Succinato-Semialdeído Desidrogenase/sangue , Succinato-Semialdeído Desidrogenase/metabolismo , Transmissão Sináptica/fisiologia , Adulto JovemRESUMO
The BCAP31 gene is located between SLC6A8, associated with X-linked creatine transporter deficiency, and ABCD1, associated with X-linked adrenoleukodystrophy. Recently, loss-of-function mutations in BCAP31 were reported in association with severe developmental delay, deafness and dystonia. We characterized the break points in eight patients with deletions of SLC6A8, BCAP31 and/or ABCD1 and studied the genotype-phenotype correlations. The phenotype in patients with contiguous gene deletions involving BCAP31 overlaps with the phenotype of isolated BCAP31 deficiency. Only deletions involving both BCAP31 and ABCD1 were associated with hepatic cholestasis and death before 1 year, which might be explained by a synergistic effect. Remarkably, a patient with an isolated deletion at the 3'-end of SLC6A8 had a similar severe phenotype as seen in BCAP31 deficiency but without deafness. This might be caused by the disturbance of a regulatory element between SLC6A8 and BCAP31.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Colestase Intra-Hepática/genética , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/mortalidade , Adrenoleucodistrofia/patologia , Adulto , Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/mortalidade , Encefalopatias Metabólicas Congênitas/patologia , Criança , Pré-Escolar , Colestase Intra-Hepática/mortalidade , Colestase Intra-Hepática/patologia , Creatina/deficiência , Creatina/genética , Deleção de Genes , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/mortalidade , Deficiência Intelectual/patologia , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/mortalidade , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Fenótipo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiênciaRESUMO
The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg(140) to Gln(140) or Gly(140) (IDH2(wt/R140Q), IDH2(wt/R140G)) in d-2-hydroxyglutaric aciduria (D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1(R132H) and IDH2(R172K) mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate (D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2(wt/R140Q) gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2(wt/R140Q) enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4-(2)H(4)]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2(wt/R140Q) enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4nmolh(-1)mgprotein(-1) vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2(wt/R140Q) activity was obtained with oxaloacetate, which competitively inhibited IDH2(wt/R140Q) activity. Lymphoblast IDH2(wt/R140Q) showed long-term cell culture stability without loss of the heterozygous IDH2(wt/R140Q) mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.
Assuntos
Encefalopatias Metabólicas Congênitas/enzimologia , Isocitrato Desidrogenase/metabolismo , Linfócitos/enzimologia , Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/terapia , Estudos de Casos e Controles , Células Cultivadas , Cromatografia Líquida , Inibidores Enzimáticos/farmacologia , Glutaratos/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Ácidos Cetoglutáricos/metabolismo , Linfócitos/efeitos dos fármacos , Linfócitos/patologia , Mutação/genética , Sensibilidade e Especificidade , Espectrometria de Massas em TandemRESUMO
BACKGROUND: GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis resulting in severe neurological complications in untreated patients. Currently available treatment is only successful to stop disease progression, but is not sufficient to reverse neurological complications occurring prior to diagnosis. Normal neurodevelopmental outcome in a patient, treated in the newborn period, highlights the importance of early diagnosis. METHODS: Targeted mutation analysis (c.59G>C and c.327G>A) in the GAMT gene by the QIAxcel system and GAA measurement by a novel two-tier method were performed in 3000 anonymized newborn blood dot spot cards. RESULTS: None of the targeted mutations were detected in any newborn. Two novel heterozygous variants (c.283_285dupGTC; p.Val95dup and c.278_283delinsCTCGATGCAC; p.Asp93AlafsX35) were identified by coincidence. Carrier frequency for these insertion/deletion types of GAMT mutations was 1/1475 in this small cohort of newborns. GAA levels were at or above the 99th percentile (3.12 µmol/l) in 4 newborns. Second-tier testing showed normal results for 4 newborns revealing 0.1% false positive rate. No GAMT mutations were identified in 4 of the newborns with elevated GAA levels in the first tier testing. CONCLUSION: This is the first two-tier study to investigate carrier frequency of GAMT deficiency in the small cohort of newborn population to establish evidence base for the first steps toward newborn screening for this treatable neurometabolic disorder.
Assuntos
Guanidinoacetato N-Metiltransferase/genética , Transtornos do Desenvolvimento da Linguagem/diagnóstico , Transtornos do Desenvolvimento da Linguagem/genética , Transtornos dos Movimentos/congênito , Adulto , Alelos , Sequência de Bases , Creatina/sangue , Análise Mutacional de DNA , Diagnóstico Precoce , Feminino , Frequência do Gene , Guanidinoacetato N-Metiltransferase/sangue , Guanidinoacetato N-Metiltransferase/deficiência , Heterozigoto , Humanos , Recém-Nascido , Transtornos do Desenvolvimento da Linguagem/sangue , Masculino , Dados de Sequência Molecular , Transtornos dos Movimentos/sangue , Transtornos dos Movimentos/diagnóstico , Transtornos dos Movimentos/genética , Mutagênese Insercional , Triagem Neonatal , Deleção de SequênciaRESUMO
The creatine transporter defect is an X-linked cause of mental retardation. We investigated the clinical features and pattern of X-inactivation in a Dutch cohort of eight female heterozygotes. We show that symptoms of the creatine transporter defect (mental retardation, learning difficulties, and constipation) can be present in female heterozygotes. We further show that the diagnosis in females is not straightforward: (i) The creatine/creatinine ratio in urine was elevated only in three of eight females. (ii) Although as a group the females had a significantly decreased cerebral creatine concentration, individual females had creatine concentrations overlapping with normal controls. (iii) Skewed X-inactivation was found in the cultured fibroblasts, in favour of either the mutated or the wild-type allele, leading to either deficient or normal results in the creatine uptake studies in fibroblasts. Thus, screening by these tests is unreliable for the diagnosis. In addition, we found no consistent skewing of the X-inactivation in peripheral tissues indicating that there is no selection against the creatine transporter defect. We conclude that testing for creatine transporter defect should be considered in females with (mild) mental retardation. Screening by DNA analysis of the SLC6A8 gene is recommended.
Assuntos
Heterozigoto , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteínas do Tecido Nervoso/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Inativação do Cromossomo X/genética , Adulto , Idoso , Células Cultivadas , Creatina/metabolismo , Feminino , Humanos , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/diagnóstico , Pessoa de Meia-Idade , Mutação , Países Baixos , Testes NeuropsicológicosRESUMO
The urinary creatine:creatinine (Cr:Crn) ratio was measured in males from 49 families with a family history compatible with X-linked mental retardation (XLMR) in order to estimate the prevalence of SLC6A8 deficiency in Estonia. We identified 11 boys from 9 families with an increased urinary Cr:Crn ratio (18%). In three related boys, a hemizygous missense mutation (c.1271G>A; p.Gly424Asp) was identified. Their mother was heterozygous for the same mutation. Although many missense mutations have been described, the p.Gly424Asp mutation has not been previously reported. The clinical expression varied widely among affected males of this family. Patients 1 and 3 had relatively mild clinical expression (mild mental retardation (MR) and attention deficit disorder), but patient 2 had all typical clinical signs of SLC6A8 defect such as moderate MR, autistic features, expressive dysphasia and epilepsy. Among our patients, we saw significant problems in speech and language development combined with attention and behavioural difficulties. The number of false-positive biochemical results with increased urinary Cr:Crn ratio was higher (18%) in our study than in previous reports (1.810%). We therefore suggest that repeated biochemical testing should be performed before DNA sequencing analysis. Our study suggests that 2% (95% confidence limits: 0.0511.1%) of this Estonian XLMR panel are due to mutations in the SLC6A8, which is similar to the prevalence reported in other populations. We therefore conclude that creatine transporter deficiency is a relatively common genetic disorder in males with sporadic or familiar MR and diagnostic screening of them should always include screening for SLC6A8 deficiency.
Assuntos
Encefalopatias Metabólicas Congênitas/diagnóstico , Creatina/deficiência , Análise Mutacional de DNA , Testes Genéticos/métodos , Deficiência Intelectual Ligada ao Cromossomo X/diagnóstico , Mutação de Sentido Incorreto , Proteínas do Tecido Nervoso/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Adolescente , Adulto , Biomarcadores/urina , Encefalopatias Metabólicas Congênitas/epidemiologia , Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/psicologia , Encefalopatias Metabólicas Congênitas/urina , Criança , Creatina/genética , Creatina/urina , Creatinina/urina , Estônia/epidemiologia , Feminino , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Humanos , Inteligência/genética , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/epidemiologia , Deficiência Intelectual Ligada ao Cromossomo X/genética , Deficiência Intelectual Ligada ao Cromossomo X/psicologia , Deficiência Intelectual Ligada ao Cromossomo X/urina , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/deficiência , Linhagem , Pessoas com Deficiência Mental , Fenótipo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/urina , Valor Preditivo dos Testes , Prevalência , Índice de Gravidade de Doença , Adulto JovemRESUMO
Malonyl coenzyme A (CoA) decarboxylase (EC 4.1.1.9, MCD) deficiency, or malonic aciduria, is a rare inborn error of metabolism characterised by a variable phenotype of developmental delay, seizures, cardiomyopathy and acidosis. There is no consensus for dietary treatment in this condition. This case describes the effect of a long-chain triglyceride (LCT)-restricted/medium-chain triglyceride (MCT)-supplemented diet upon the progress of an affected child. A full-term Asian girl of birth weight 3590 g was screened for malonic aciduria after birth due to a positive family history. She had elevated urine malonic and methylmalonic acids and was presumably homozygous for a deleterious mutation in the MLYCD gene. Her echocardiography showed mild cardiomyopathy at 0.5 months of age, but heart function was good. She was treated with carnitine 100 mg/kg per day and continued a high-energy formula feed, as her growth was slow. At 3 months of age, echocardiography showed deteriorating cardiac function with a fractional shortening of 18%. She started an angiotensin-converting enzyme (ACE) inhibitor (Captopril). Over the next few months, her diet was altered to comprise 1.9% energy from LCT, 25% from MCT and the remainder carbohydrate. Cardiac function improved and was optimal at 23 months of age, with a fractional shortening of 28% and good systolic function. During a period of low MCT intake, her cardiac function was noted to deteriorate. This reversed and stabilised following reinstatement of the diet. This case of malonic aciduria with cardiomyopathy demonstrates improvement in cardiac function attributable to LCT-restricted/MCT-supplemented diet.
Assuntos
Carboxiliases/deficiência , Cardiomiopatias/dietoterapia , Suplementos Nutricionais , Erros Inatos do Metabolismo/dietoterapia , Triglicerídeos/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Captopril , Carboxiliases/genética , Cardiomiopatias/diagnóstico , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/enzimologia , Cardiomiopatias/genética , Carnitina/uso terapêutico , Pré-Escolar , Terapia Combinada , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Fórmulas Infantis , Fenômenos Fisiológicos da Nutrição do Lactente , Recém-Nascido , Malonil Coenzima A/genética , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/enzimologia , Erros Inatos do Metabolismo/genética , Ácido Metilmalônico , Mutação , Estado Nutricional , Fenótipo , Resultado do TratamentoRESUMO
D: -2-Hydroxyglutaric aciduria (D: -2-HGA) is a neurometabolic disorder characterized by elevated levels of D: -2-hydroxyglutarate (D: -2-HG) in physiological fluids. Recent findings revealed that mutations in the D2HGDH gene, encoding D: -2-hydroxyglutarate dehydrogenase, cause D: -2-HGA. So far, a functionalenzyme assay to determine D: -2-hydroxyglutarate dehydrogenase activity, converting D: -2-HG into 2-ketoglutarate (2-KG), has been unavailable. We have now developed a unique enzyme assay for the determination of D: -2-hydroxyglutarate dehydrogenase activity in cells derived from D: -2-HGA patients and controls. The enzyme assay was performed using enantiomerically pure stable-isotope-labelled D: -2-hydroxy[3,3,4,4-(2)H(4)]glutarate. This substrate is convertedby D: -2-hydroxyglutarate dehydrogenase into 2-[3,3,4,4-(2)H(4)]ketoglutarate, which is subsequently converted into L: -[3,3,4,4-(2)H(4)]glutamate by L: -glutamate dehydrogenase, present in saturating amounts in cell homogenates. Enzyme activities were quantified using LC-MS/MS. The mean activities in control fibroblast and lymphoblast homogenates were 298 +/- 207 and 1670 +/- 940 pmol/h per mg protein, respectively. In fibroblast and lymphoblast cell lines derived from patients with pathogenic mutations in the D2HGDH gene, considerably decreased enzyme activities (e.g. <41 pmol/h per mg protein) were found compared with controls. This enzyme assay will have additional utility in further differentiating patients with D: -2-HGA and L: -2-HGA and in assessing the residual activities linked to pathogenic mutations in the D2HGDH gene.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/enzimologia , Glutaratos/urina , Algoritmos , Cromatografia Líquida de Alta Pressão , Fibroblastos/enzimologia , Glutaratos/isolamento & purificação , Meia-Vida , Humanos , Linfócitos/enzimologia , Espectrometria de Massas , EstereoisomerismoRESUMO
L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an L-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled L-2-hydroxyglutarate to 2-ketoglutarate, which is converted into L-glutamate in situ. The formation of stable isotope labelled L-glutamate is therefore a direct measure of L-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of L-2-HGDH activity was detected in cell lysates from 15 out of 15 L-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one L-2-HGA patient. The L-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue L-2-HGDH activity.
Assuntos
Oxirredutases do Álcool/análise , Oxirredutases do Álcool/deficiência , Encefalopatias Metabólicas Congênitas/diagnóstico , Extratos Celulares/química , Ensaios Enzimáticos/métodos , Oxirredutases do Álcool/líquido cefalorraquidiano , Animais , Encefalopatias Metabólicas Congênitas/líquido cefalorraquidiano , Encefalopatias Metabólicas Congênitas/patologia , Calibragem , Extratos Celulares/análise , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida/métodos , Ensaios Enzimáticos/normas , Fibroblastos/química , Fibroblastos/enzimologia , Humanos , Linfócitos/química , Linfócitos/enzimologia , Modelos Biológicos , Modelos Moleculares , Ratos , Projetos de Pesquisa , Espectrometria de Massas em Tandem/métodosRESUMO
BACKGROUND: Treatment with oral creatine monohydrate has not shown efficacy in patients with creatine transporter deficiency (CRTR-D). Another therapeutic option proposed is L-arginine, the substrate for the enzyme L-arginine:glycine amidinotransferase (AGAT). We evaluate clinical characteristics and cerebral creatine replenishment after L-arginine therapy in four patients with CRTR-D. PATIENTS AND METHODS: Four boys with genetically confirmed diagnosis of CRTR-D (ages 9-16 years) were supplemented with L-arginine (0.4 g/kg per day) for a period of 9 months. Treatment efficacy was evaluated by clinical and neuropsychological assessment and determination of creatine signals by brain proton magnetic resonance spectroscopy ((1)H-MRS). RESULTS: Epileptic seizures remained well controlled with antiepileptic drugs in three cases, both before and after L-arginine supplementation. Vineland Adaptive Behaviour Scale did not show any change in communication, daily living skills, socialization or motor skills, and a lack of improvement in brain (1)H-MRS follow-up was observed. L-Arginine was discontinued at the end of the observation period. CONCLUSIONS: Nine months of L-arginine supplementation did not show effectiveness in the four patients affected with CRTR-D in this protocol.
Assuntos
Arginina/uso terapêutico , Genes Ligados ao Cromossomo X , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Administração Oral , Adolescente , Idade de Início , Criança , Cromossomos Humanos X/genética , Creatina/administração & dosagem , Suplementos Nutricionais , Epilepsia/tratamento farmacológico , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/tratamento farmacológico , Erros Inatos do Metabolismo/genéticaRESUMO
In November 2007 Varian Medical Systems released a new version of ECLIPSE (version 8.1.17), which includes a correction to its Anisotropic Analytical Algorithm affecting monitor unit calculations for Enhanced Dynamic Wedge. The purpose of this study is to evaluate and compare the accuracy of the MU calculations for version 8.1.17 with that from the previous version (8.0.05).
Assuntos
Algoritmos , Radiometria/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Radioterapia Conformacional/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , SoftwareRESUMO
L-2-Hydroxyglutaric aciduria (L-2-HGA) is an autosomal recessive neurometabolic disorder characterized by psychomotor delay, ataxia, macrocephaly and typical neuroradiological findings of subcortical leucoencephalopathy. Recently, the disease causing gene has been discovered (L2HGDH) encoding L-2-hydroxyglutarate dehydrogenase. We present a 3-year-old boy with L-2-HGA, who demonstrated macrocephaly, noted already in utero with ultrasound. Cranial MRI demonstrated diffuse subcortical encephalopathy with increased signal of the subcortical white matter. Subsequent metabolic screening revealed increased levels of L-2-HGA, and genomic DNA analysis demonstrated two missense mutations in L-2-HGDG. Patient's further motor development was mildly impaired, whilst his speech development was profoundly impaired (first words at the age of 2 years). Since the age of 2 years he started demonstrating autistic repetitive behaviors and movements, increasing aloofness to his environment and limitations in the variety of spontaneous activity (CARS score: 44/60-severe autism). Autism has not so far been described in L-2-HGA and may be considered as an additional feature of the phenotypic spectrum.
Assuntos
Oxirredutases do Álcool/genética , Erros Inatos do Metabolismo dos Aminoácidos/complicações , Erros Inatos do Metabolismo dos Aminoácidos/genética , Transtorno Autístico/etiologia , Transtorno Autístico/genética , Glutaratos/urina , Erros Inatos do Metabolismo dos Aminoácidos/urina , Transtorno Autístico/urina , Encéfalo/patologia , Humanos , Lactente , Imageamento por Ressonância Magnética , Masculino , Fenótipo , Índice de Gravidade de DoençaRESUMO
Guanidinoacetate methyltransferase (GAMT) deficiency is a rare disorder of creatine synthesis. We report a patient who presented at 10 months of age with hypotonia and global developmental delay. Subsequently, she developed seizures and choreoathetosis. Magnetic resonance imaging showed high signal bilaterally in the globus pallidus on T2-weighted images. Mitochondrial respiratory chain studies revealed low complex I activity (in muscle 0.052 nmol NADH oxidized per min per unit citrate synthase, controls 0.166 +/- 0.047; in fibroblasts 0.080 nmol NADH oxidized per min per unit citrate synthase, controls 0.197 +/- 0.034). The true diagnosis was suspected at 21 months of age because of persistent low plasma and urine creatinine concentrations. GAMT activity was undetectable in fibroblasts and compound heterozygous mutations were found in the GAMT gene (c.327G>A and c.522G>A). The patient was treated with creatine, dietary arginine restriction and ornithine supplements. Her movement disorder and seizures resolved but she still has severe cognitive impairment and no expressive language. The occurrence of secondary respiratory chain abnormalities in GAMT deficiency may lead to misdiagnosis, particularly as the clinical and radiological features resemble those seen in mitochondrial encephalopathies. It is important to establish the correct diagnosis because specific treatment is available.
Assuntos
Encefalopatias/diagnóstico , Guanidinoacetato N-Metiltransferase/deficiência , Mitocôndrias/patologia , Encéfalo/patologia , Diagnóstico Diferencial , Feminino , Fibroblastos/metabolismo , Heterozigoto , Humanos , Lactente , Imageamento por Ressonância Magnética , MutaçãoRESUMO
We report nine new patients with malonic aciduria associated with enzyme-confirmed malonyl-CoA decarboxylase (MCD) deficiency in eight. Clinical details were available on eight, and molecular genetic characterization was obtained for nine. As for 15 previously described patients, cardinal clinical manifestations included developmental delay and cardiomyopathy; metabolic perturbations (e.g. acidosis) and seizures, however, were infrequent or not observed in our patients. For all, detection of elevated malonic acid in urine (+/- increased C3DC acylcarnitine by analysis employing tandem mass spectrometry) led to pursuit of enzyme studies. MCD activities (nmol/h PER mg protein) revealed: control (n = 22), 16.2 +/- 1.8 (SEM; range 5.7-46.2); patients (n = 8, assayed in duplicate), 1.7 +/- 0.3 (10% of parallel control; range 0.6-2.8). Molecular characterization by DNA sequence analysis and multiplex ligation-dependent probe amplification revealed nine novel mutations (c.796C>T; p.Gln266X, c.481delC; p.Leu161CysfsX18, c.1367A>C; p.Tyr456Ser, c.1319G>T; p.Ser440Ile, c.1430C>T; p.Ser477Phe, c.899G>T; p.Gly300Val, c.799-1683_949-1293del3128, and two other large genomic deletions comprising exons 1 or the complete gene) and two known mutations in the MLYCD gene. Our findings increase the number of enzyme-confirmed MCD-deficient patients by >50%, and expand our understanding of the phenotypic and molecular heterogeneity of this rare disorder.
Assuntos
Carboxiliases/deficiência , Carboxiliases/genética , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/genética , Mutação , Adolescente , Criança , Pré-Escolar , Éxons , Feminino , Deleção de Genes , Humanos , Lactente , Masculino , Malonatos/urina , Erros Inatos do Metabolismo/sangue , Modelos Biológicos , Modelos Genéticos , Fenótipo , Reação em Cadeia da PolimeraseRESUMO
HYPOTHESIS: An enzymatic assay for quantification of γ-hydroxybutyric acid (GHB) in biofluids can be employed for targeted screening of succinic semialdehyde dehydrogenase deficiency (SSADHD) in selected populations. RATIONALE: We used a two-tiered study approach, in which the first study (proof of concept) examined 7 urine samples derived from patients with SSADHD and 5 controls, and the second study (feasibility study) examined a broader sample population of patients and controls, including plasma. OBJECTIVE: Split samples of urine and plasma (anonymized) were evaluated by enzymatic assay, gas chromatography alone (proof of concept) and gas chromatography-mass spectrometry, and the results compared. METHOD: Multiple detection methods have been developed to detect GHB. We evaluated an enzymatic assay which employs recombinant GHB dehydrogenase coupled to NADH production, the latter quantified on a Cobas Integra 400 Plus. Results: In our proof of concept study, we analyzed 12 urine samples (5 controls, 7 SSADHD), and in the feasibility study we evaluated 33 urine samples (23 controls, 10 SSADHD) and 31 plasma samples (14 controls, 17 SSADHD). The enzymatic assay carried out on a routine clinical chemistry analyzer was robust, revealing excellent agreement with instrumental methods in urine (GC-FID: r = 0.997, p ≤ 0.001; GC-MS: r = 0.99, p ≤ 0.001); however, the assay slightly over-estimated GHB levels in plasma, especially those in which GHB levels were low. Conversely, correlations for the enzymatic assay with comparator methods for higher plasma GHB levels were excellent (GC-MS; r = 0.993, p ≤ 0.001). CONCLUSION: We have evaluated the capacity of this enzymatic assay to identify patients with SSADHD via quantitation of GHB. The data suggests that the enzymatic assay may be a suitable screening method to detect SSADHD in selected populations using urine. In addition, the assay can be used in basic research the elucidate the mechanism of the underlying disease or monitor GHB- levels for the evaluation of drug candidates. SYNOPSIS: An enzymatic assay for GHB in biofluids was evaluated as a screening method for SSADHD and found to be reliable in urine, but in need of refinement for application to plasma.
RESUMO
BACKGROUND: GAMT deficiency is an autosomal recessive disorder of creatine biosynthesis causing developmental delays or intellectual disability in untreated patients as a result of irreversible brain damage occurring prior to diagnosis. Normal neurodevelopmental outcome has been reported in patients treated from neonatal period highlighting the importance of early treatment. METHODS: Five hundred anonymized newborns from the National Newborn Screening Program of The Netherlands were included into this pilot study. Direct sequencing of the coding region of the GAMT gene was applied following DNA extraction. The disease causing nature of novel missense variants in the GAMT gene was studied by overexpression studies. GAA and creatine was measured in blood dot spots. RESULTS: We detected two carriers, one with a known common (c.327G>A) and one with a novel mutation (c.297_309dup (p.Arg105Glyfs*) in the GAMT gene. The estimated incidence of GAMT deficiency was 1:250,000. We also detected five novel missense variants. Overexpression of these variants in GAMT deficient fibroblasts did restore GAMT activity and thus all were considered rare, but not disease causing variants including the c.131G>T (p.Arg44Leu) variant. Interestingly, this variant was predicted to be pathogenic by in silico analysis. The variants were included in the Leiden Open Variation Database (LOVD) database (www.LOVD.nl/GAMT). The average GAA level was 1.14µmol/L±0.45 standard deviations. The average creatine level was 408µmol/L±106. The average GAA/creatine ratio was 2.94±0.136. CONCLUSION: The estimated incidence of GAMT deficiency is 1:250,000 newborns based on our pilot study. The newborn screening for GAMT deficiency should be implemented to identify patients at the asymptomatic stage to achieve normal neurodevelopmental outcome for this treatable neurometabolic disease. Biochemical investigations including GAA, creatine and GAMT enzyme activity measurements are essential to confirm the diagnosis of GAMT deficiency. According to availability, all missense variants can be assessed functionally, as in silico prediction analysis of missense variants is not sufficient to confirm the pathogenicity of missense variants.
Assuntos
Bases de Dados de Ácidos Nucleicos , Guanidinoacetato N-Metiltransferase/deficiência , Transtornos do Desenvolvimento da Linguagem/genética , Transtornos dos Movimentos/congênito , Feminino , Guanidinoacetato N-Metiltransferase/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Incidência , Recém-Nascido , Transtornos do Desenvolvimento da Linguagem/epidemiologia , Masculino , Programas de Rastreamento , Transtornos dos Movimentos/epidemiologia , Transtornos dos Movimentos/genética , Mutação de Sentido Incorreto , Projetos PilotoRESUMO
We report a patient with anti-epileptic treatment refractory neonatal seizures responsive to pyridoxine. Biochemical analysis revealed normal markers for antiquitin deficiency and also mutation analysis of the ALDH7A1 (Antiquitin) gene was negative. Mutation analysis of the PNPO gene revealed a novel, homozygous, presumed pathogenic mutation (c.481C > T; p.(Arg161Cys)). Measurements of B6 vitamers in a CSF sample after pyridoxine administration revealed elevated pyridoxamine as the only metabolic marker for PNPO deficiency. With pyridoxine monotherapy the patient is seizure free and neurodevelopmental outcome at the age of 14 months is normal.
RESUMO
We studied 21 patients, from 18 families, with L-2-hydroxyglutaric aciduria (L-2-HGA), a rare neurometabolic disorder with a homogeneous presentation: progressive neurodegeneration with extrapyramidal and cerebellar signs, seizures, and subcortical leukoencephalopathy. Increased levels of L-2-hydroxyglutaric acid in body fluids proved the diagnosis of L-2-HGA in all 21 patients. We analyzed the L-2-HGA gene (L2HGDH), recently found to be mutated in consanguineous families with L-2-HGA, and identified seven novel mutations in 15 families. Three mutations appeared to be particularly prevalent in this Portuguese panel: a frameshift mutation (c.529delC) was detected in 12 out of 30 mutant alleles (40%), a nonsense mutation (c.208C>T; p.Arg70X) in 7/30 alleles (23%), and a missense mutation (c.293A>G; p.His98Arg) in four out of 30 mutant alleles (13%), suggesting that common origin may exist. Furthermore, two novel missense (c.169G>A; p.Gly57Arg, c.1301A>C; p.His434Pro) and two splice error (c.257-2A>G, c.907-2A>G) mutations were found. All the mutations presumably lead to loss-of-function with no relationship between clinical signs, progression of the disease, levels of L-2-HGA and site of the mutation. In the three remaining families, no pathogenic mutations in the L-2-HGA were found, which suggests either alterations in regulatory regions of the gene or of its intervening sequences, compound heterozygosity for large genomic deletion and, or further genetic heterogeneity.
Assuntos
Oxirredutases do Álcool/genética , Glutaratos/urina , Mutação , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Estudos de Coortes , Consanguinidade , Saúde da Família , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Portugal , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Glucocorticoid (GC) hormones induce apoptosis in lymphoid and leukemic cells by binding and activating cytosolic GC receptors. Because physiological stress often causes hormone-free GC receptor activation, we have investigated if stress-induced apoptosis of lymphoid cells is also mediated by the activation of the GC receptor pathway. In S49 T lymphoma cells, heat shock and deprivation of growth factors or nutrients caused nuclear translocation and loss of agonist binding capacity of GC receptors, similar to that in cells incubated with the glucocorticoid dexamethasone (DEX). In variant S49 H.2 cells, cross-resistance to DEX, temperature shocks and growth factor deprivation were associated with a higher threshold for hormone-dependent and -independent receptor activation in situ and with impaired in vitro activation of cytosolic receptors. Cross-resistance to DEX, low serum and heat shock was abrogated, however, by pharmacological sensitization of GC receptor activation with the drug meta-iodobenzylguanidine (MIBG). Sensitive S49 cells and resistant variants did not differ in the expression levels of the apoptosis-regulating genes bax, bad, bcl-X and bcl-2, the status of the p53 gene nor in a different requirement for the growth factors II-2, IL-4 or IL-9. The results suggest that ligand-independent activation of the GC receptor is a central signalling and controlling event in some forms of stress-induced apoptosis, assigning a novel function to the GC receptor in the regulation of lymphoid and leukemic cell numbers.