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Glutaric Aciduria type I (GA1) is a rare neurometabolic disorder caused by mutations in the GDCH gene encoding for glutaryl-CoA dehydrogenase (GCDH) in the catabolic pathway of lysine, hydroxylysine and tryptophan. GCDH deficiency leads to increased concentrations of glutaric acid (GA) and 3-hydroxyglutaric acid (3-OHGA) in body fluids and tissues. These metabolites are the main triggers of brain damage. Mechanistic studies supporting neurotoxicity in mouse models have been conducted. However, the different vulnerability to some stressors between mouse and human brain cells reveals the need to have a reliable human neuronal model to study GA1 pathogenesis. In the present work we generated a GCDH knockout (KO) in the human neuroblastoma cell line SH-SY5Y by CRISPR/Cas9 technology. SH-SY5Y-GCDH KO cells accumulate GA, 3-OHGA, and glutarylcarnitine when exposed to lysine overload. GA or lysine treatment triggered neuronal damage in GCDH deficient cells. SH-SY5Y-GCDH KO cells also displayed features of GA1 pathogenesis such as increased oxidative stress vulnerability. Restoration of the GCDH activity by gene replacement rescued neuronal alterations. Thus, our findings provide a human neuronal cellular model of GA1 to study this disease and show the potential of gene therapy to rescue GCDH deficiency.
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Erros Inatos do Metabolismo dos Aminoácidos , Encefalopatias Metabólicas , Lisina , Neuroblastoma , Humanos , Animais , Camundongos , Lisina/genética , Glutaril-CoA Desidrogenase/genética , Glutaril-CoA Desidrogenase/metabolismo , Camundongos Knockout , Terapia GenéticaRESUMO
Short-chain enoyl-CoA hydratase (ECHS1) is a mitochondrial beta-oxidation enzyme involved in the metabolism of acyl-CoA fatty acid esters, as well as in valine metabolism. ECHS1 deficiency has multiple manifestations, including Leigh syndrome early at birth or in childhood with poor prognosis, to cutis laxa, exercise-induced dystonia and congenital lactic acidosis. Here we describe the case of a newborn with mutations in ECHS1 that caught our attention after the incidental finding of 3-hydroxy-butyryl\3-hydroxy-isobutyryl\malonylcarnitine (C4OH\C3DC) and tiglylcarnitine (C5:1) on blood spot in the newborn screening (NBS) program. Diagnosis was suspected based on the analysis of organic acids on dried urine spot. A moderate increase of 2-methyl-2,3-dihydroxybutyric acid, was detected, which is a known marker of this disease. Exome analysis showed c.404A>G (p.Asn135Ser) mutation in homozygosis in the ECHS1 gene. The child was therefore admitted to the hospital. Initial examination showed little response to auditory stimuli and mild hypertonia of the extremities. Clinical deterioration was evident at 4 months of age, including neurological and cardiac involvement, and the patient died at 5 months of age. This case illustrates how an incidental detection in the NBS Program can lead to the diagnosis ECHS1 deficiency. Although it is a severe disease, with no treatment available, early detection would allow adequate genetic counseling avoiding the odyssey that suffered most of these families.
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Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetic disorders, all of which impair neuromuscular transmission. Epidemiological data and frequencies of gene mutations are scarce in the literature. Here we describe the molecular genetic and clinical findings of sixty-four genetically confirmed CMS patients from Spain. Thirty-six mutations in the CHRNE, RAPSN, COLQ, GFPT1, DOK7, CHRNG, GMPPB, CHAT, CHRNA1, and CHRNB1 genes were identified in our patients, with five of them not reported so far. These data provide an overview on the relative frequencies of the different CMS subtypes in a large Spanish population. CHRNE mutations are the most common cause of CMS in Spain, accounting for 27% of the total. The second most common are RAPSN mutations. We found a higher rate of GFPT1 mutations in comparison with other populations. Remarkably, several founder mutations made a large contribution to CMS in Spain: RAPSN c.264C > A (p.Asn88Lys), CHRNE c.130insG (Glu44Glyfs*3), CHRNE c.1353insG (p.Asn542Gluf*4), DOK7 c.1124_1127dup (p.Ala378Serfs*30), and particularly frequent in Spain in comparison with other populations, COLQ c.1289A > C (p.Tyr430Ser). Furthermore, we describe phenotypes and distinguishing clinical signs associated with the various CMS genes which might help to identify specific CMS subtypes to guide diagnosis and management.
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Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/fisiopatologia , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Miastênicas Congênitas/classificação , Síndromes Miastênicas Congênitas/epidemiologia , Espanha/epidemiologia , Adulto JovemRESUMO
INTRODUCTION: Spasticity is a medical problem with a high incidence that significantly impact on the quality of life of patients and their families. AIM: To analyze and to answer different questions about the use of botulinum toxin type A (BTA) in our clinical practice. DEVELOPMENT: A group of experts in neurology develop a list of topics related with the use of BTA. Two big groups were considered: spasticity in adults and in children with cerebral palsy. A literature search at PubMed for English, French, and Spanish language articles published up to June 2016 was performed. The manuscript was structured as a questionnaire that includes those questions that, according to the panel opinion, could generate more controversy or doubt. The initial draft was reviewed by the expert panel members to allow for modifications, and after subsequent revisions for achieving the highest degree of consensus, the final text was then validated. Different questions about diverse aspects of spasticity in adults, such as methods for evaluating spasticity, infiltration techniques, doses, number of infiltration points, etc. Regarding spasticity in children with cerebral palsy, the document included questions about minimum age of infiltration, methods of analgesia, etc. CONCLUSIONS: This review is a tool for continuous training for neurologist and rehabilitation specialist and residents of both specialties, about different specific areas of the management of BTA.
TITLE: Mitos y evidencias en el empleo de la toxina botulinica: espasticidad del adulto y del nintilde;o con paralisis cerebral.Introduccion. La espasticidad es un problema medico frecuente que impacta de forma significativa en la calidad de vida de los pacientes y sus familias. Objetivo. Analizar y dar respuesta a diferentes cuestiones en el uso de la toxina botulinica tipo A (TBA) en nuestra practica clinica habitual. Desarrollo. Un grupo de expertos en neurologia elaboro una lista de temas relacionados con el uso de la TBA. Se consideraron dos grandes bloques: espasticidad del adulto y del nintilde;o con paralisis cerebral. Se realizo una revision de la bibliografia que incluyo los diferentes articulos publicados en espantilde;ol, ingles y frances hasta junio de 2016. El documento se estructuro como un cuestionario que incluyo las preguntas que, segun el criterio del panel, podrian generar mayor controversia o duda. El borrador inicial del documento fue revisado por los miembros del panel y se realizaron las modificaciones necesarias hasta alcanzar el mayor grado de consenso. A continuacion, el texto final fue validado. Se incluyeron diferentes preguntas sobre diferentes aspectos de la espasticidad en adultos: evaluacion de la espasticidad, tecnicas de infiltracion, dosis, numero de puntos, etc. En cuanto a la espasticidad en los nintilde;os con paralisis cerebral, se analizaron preguntas como: edad minima de infiltracion, metodos de sedoanalgesia, etc. Conclusiones. Esta revision constituye una herramienta para neurologos, medicos rehabilitadores y residentes de ambas especialidades, dentro de diferentes ambitos especificos del manejo de la TBA.
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Toxinas Botulínicas Tipo A/uso terapêutico , Paralisia Cerebral/tratamento farmacológico , Fármacos Neuromusculares/uso terapêutico , Adolescente , Adulto , Toxinas Botulínicas Tipo A/administração & dosagem , Toxinas Botulínicas Tipo A/efeitos adversos , Paralisia Cerebral/reabilitação , Paralisia Cerebral/terapia , Criança , Pré-Escolar , Terapia Combinada , Consenso , Gerenciamento Clínico , Feminino , Objetivos , Humanos , Lactente , Masculino , Esclerose Múltipla/complicações , Esclerose Múltipla/tratamento farmacológico , Espasticidade Muscular/tratamento farmacológico , Espasticidade Muscular/terapia , Fármacos Neuromusculares/administração & dosagem , Fármacos Neuromusculares/efeitos adversos , Modalidades de Fisioterapia , Inquéritos e Questionários , Avaliação de Sintomas , Adulto JovemRESUMO
Multiple myeloma (MM) is a plasma cell malignancy where MM cell growth is supported by the bone marrow (BM) microenvironment with poorly defined cellular and molecular mechanisms. MM cells express CD40, a receptor known to activate autocrine secretion of cytokines and elicit proliferation. Activated T helper (Th) cells express CD40 ligand (CD40L) and BM Th cells are significantly increased in MM patients. We hypothesized that activated BM Th cells could support MM cell growth. We here found that activated autologous BM Th cells supported MM cell growth in a contact- and CD40L-dependent manner in vitro. MM cells had retained the ability to activate Th cells that reciprocated and stimulated MM cell proliferation. Autologous BM Th cells supported MM cell growth in xenografted mice and were found in close contact with MM cells. MM cells secreted chemokines that attracted Th cells, secretion was augmented by CD40-stimulation. Within 14 days of culture of whole BM aspirates in autologous serum, MM cells and Th cells mutually stimulated each other, and MM cells required Th cells for further expansion in vitro and in mice. The results suggest that Th cells may support the expansion of MM cells in patients.
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Transplante de Medula Óssea/efeitos adversos , Mieloma Múltiplo/patologia , Linfócitos T Auxiliares-Indutores/transplante , Evasão Tumoral/imunologia , Idoso , Animais , Apresentação de Antígeno , Antígenos CD40/imunologia , Ligante de CD40/imunologia , Divisão Celular , Quimiocinas/metabolismo , Quimiotaxia de Leucócito , Técnicas de Cocultura , Citocinas/metabolismo , Sobrevivência de Enxerto/imunologia , Xenoenxertos , Humanos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/terapia , Linfócitos T Auxiliares-Indutores/imunologia , Transplante Autólogo/efeitos adversos , Microambiente TumoralRESUMO
The analysis of acylcarnitines (AC) in plasma/serum is established as a useful test for the biochemical diagnosis and the monitoring of treatment of organic acidurias and fatty acid oxidation defects. External quality assurance (EQA) for qualitative and quantitative AC is offered by ERNDIM and CDC in dried blood spots but not in plasma/serum samples. A pilot interlaboratory comparison between 14 European laboratories was performed over 3 years using serum/plasma samples from patients with an established diagnosis of an organic aciduria or fatty acid oxidation defect. Twenty-three different samples with a short clinical description were circulated. Participants were asked to specify the method used to analyze diagnostic AC, to give quantitative data for diagnostic AC with the corresponding reference values, possible diagnosis, and advice for further investigations.Although the reference and pathological concentrations of AC varied among laboratories, elevated marker AC for propionic acidemia, isovaleric acidemia, medium-chain acyl-CoA dehydrogenase, very long-chain acyl-CoA dehydrogenase, and multiple acyl-CoA dehydrogenase deficiencies were correctly identified by all participants allowing the diagnosis of these diseases. Conversely, the increased concentrations of dicarboxylic AC were not always identified, and therefore the correct diagnosis was not reach by some participants, as exemplified in cases of malonic aciduria and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency. Misinterpretation occurred in those laboratories that used multiple-reaction monitoring acquisition mode, did not derivatize, or did not separate isomers. However, some of these laboratories suggested further analyses to clarify the diagnosis.This pilot experience highlights the importance of an EQA scheme for AC in plasma.
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Rapsyn (RAPSN) mutations are a common cause of postsynaptic congenital myasthenic syndromes. We present a comprehensive description of the clinical and molecular findings of ten patients with CMS due to mutations in RAPSN, mostly with a long-term follow-up. Two patients were homozygous and eight were heterozygous for the common p.Asn88Lys mutation. In three of the heterozygous patients we have identified three novel mutations (c.869T > C; p.Leu290Pro, c.1185delG; p.Thr396Profs*12, and c.358delC; p.Gln120Serfs*8). In our cohort, the RAPSN mutations lead to a relatively homogeneous phenotype, characterized by fluctuating ptosis, occasional bulbar symptoms, neck muscle weakness, and mild proximal muscle weakness with exacerbations precipitated by minor infections. Interestingly, episodic exacerbations continue to occur during adulthood. These were characterized by proximal limb girdle weakness and ptosis, and not so much by respiratory insufficiency after age 6. All patients presented during neonatal period and responded to cholinergic agonists. In most of the affected patients, additional use of 3,4-diaminopyridine resulted in significant clinical benefit. The disease course is stable except for intermittent worsening.
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Inibidores da Colinesterase/farmacologia , Progressão da Doença , Proteínas Musculares/genética , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/fisiopatologia , Bloqueadores dos Canais de Potássio/farmacologia , 4-Aminopiridina/administração & dosagem , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/farmacologia , Adolescente , Adulto , Amifampridina , Criança , Pré-Escolar , Inibidores da Colinesterase/administração & dosagem , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Síndromes Miastênicas Congênitas/tratamento farmacológico , Fenótipo , Bloqueadores dos Canais de Potássio/administração & dosagem , Brometo de Piridostigmina/administração & dosagem , Brometo de Piridostigmina/farmacologia , Adulto JovemRESUMO
Laboratory data interpretation for the assessment of complex biological systems remains a great challenge, as occurs in mitochondrial function research studies. The classical biochemical data interpretation of patients versus reference values may be insufficient, and in fact the current classifications of mitochondrial patients are still done on basis of probability criteria. We have developed and applied a mathematic agglomerative algorithm to search for correlations among the different biochemical variables of the mitochondrial respiratory chain in order to identify populations displaying correlation coefficients >0.95. We demonstrated that coenzyme Q10 may be a better biomarker of mitochondrial respiratory chain enzyme activities than the citrate synthase activity. Furthermore, the application of this algorithm may be useful to re-classify mitochondrial patients or to explore associations among other biochemical variables from different biological systems.
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Algoritmos , Citrato (si)-Sintase/análise , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias Musculares/enzimologia , Ubiquinona/análogos & derivados , Adolescente , Biomarcadores/análise , Criança , Pré-Escolar , Transporte de Elétrons , Humanos , Lactente , Doenças Mitocondriais/enzimologia , Ubiquinona/análiseRESUMO
Glutaric acidemia type I (GA-I) is a treatable autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism caused by glutaryl-CoA dehydrogenase (GCDH) deficiency. Presentation and progression of disease are variable ranging from asymptomatic carrier state to catastrophic encephalopathy. GA-I usually presents before age 18 months, usually triggered by childhood infection, with mild or severe acute encephalopathy, striatal degeneration, and movement disorder, most often acute dystonia. At a presymptomatic stage diagnosis is suggested clinically by macrocephaly, radiologically by widened Sylvian fissures and biochemically by the presence of excess 3-hydroxyglutaric acid and glutaric acid in urine. Treatment consists of lysine-restricted diet and carnitine supplementation, specific diet restrictions, as well as symptomatic and anticatabolic treatment of intercurrent illness. Presymptomatic diagnosis and treatment are essential to prognosis. We report the case of 16-year-old macrocephalic female with late-onset GA-I and unusual paucisymptomatic presentation with fainting after exercise and widespread white matter signal changes at MRI. She was compound heterozygote for a novel mutation (IVS10-2A>G) affecting splicing at GCDH and a common missense mutation (c. 1240C>T; p.Arg402Trp, R402W). Interestingly, the site of the novel mutation is the nucleotide position of a common mutation found almost exclusively in patients of Chinese/Taiwanese origin (IVS10-2A>C).
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BACKGROUND: CblC deficiency produces a combination of methylmalonic aciduria (MMA) and homocystinuria (HCU), and is the most common error of cobalamin metabolism. Patients present a wide spectrum of symptoms, ranging from early severe multisystemic forms, to milder late-onset phenotypes. Cognitive and visual impairment are nearly constant. Hydroxocobalamin (OHCbl), betaine, folinic acid, levocarnitine and eventually dietary protein restriction are the main therapeutic approaches. Although early introduction of OHCbl is crucial, no standardized protocols regarding dose adaptation exist. No reports on long-term outcomes after high doses of this vitamin have been published. METHODS: In this study five patients with CblC deficiency (early severe forms) were treated with high doses of OHCbl for 18 to 30months. Clinical examinations, neurological assessment, and biochemical studies (plasma total homocysteine (tHcy), amino acids, hydroxocobalamin, and methylmalonic acid in urine) were periodically performed. RESULTS: Variable clinical and biochemical outcomes were observed in patients treated with high doses of OHCbl. The best biochemical response was observed in those children with the worse metabolic control. By contrast, those patients with a concentration of tHcy around 50µmol/l or less showed only minor changes. Clinically, a considerable improvement was observed in those patients with severe problems in communication, expressive language and behavior. CONCLUSIONS: According to our study, high OHCbl doses in CblC deficiency could have a greater benefit in those children with a prior history of suboptimal metabolic control, and also in those with severe neurological phenotypes. More specifically, we observed improvements in communication skills and behavior. These results should encourage further prospective trials to determine the optimal OHCbl regimen and to generate protocols and guidelines in this rare disorder.
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Hidroxocobalamina/administração & dosagem , Deficiência de Vitamina B 12/tratamento farmacológico , Vitamina B 12/metabolismo , Idade de Início , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Feminino , Homocistinúria/diagnóstico , Humanos , Masculino , Resultado do Tratamento , Deficiência de Vitamina B 12/patologiaRESUMO
BACKGROUND: Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. Since its first description in 2001 several case reports have been published but an overview of phenotype, genotype and phenotype--genotype correlation has been lacking. METHODS: We performed a retrospective study of clinical, biochemical and molecular genetic data of 101 males with X-linked creatine transporter deficiency from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). RESULTS AND CONCLUSIONS: Most patients developed moderate to severe intellectual disability; mild intellectual disability was rare in adult patients. Speech language development was especially delayed but almost a third of the patients were able to speak in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine to creatinine ratio proved to be a reliable screening method besides MR spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. A third of patients had a de novo mutation in the SLC6A8 gene. Mothers with an affected son with a de novo mutation should be counselled about a recurrence risk in further pregnancies due to the possibility of low level somatic or germline mosaicism. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesise creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurones.
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Encefalopatias Metabólicas Congênitas/genética , Creatina/deficiência , Creatina/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/genética , Proteínas do Tecido Nervoso/genética , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/deficiência , Adulto , Criança , Creatina/genética , Genes Ligados ao Cromossomo X , Testes Genéticos , Genótipo , Humanos , Masculino , Fenótipo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/genética , Estudos RetrospectivosRESUMO
Coenzyme Q(10) (CoQ(10)) deficiency syndromes are potentially treatable disorders. Skeletal muscle is the most widely accepted tissue for their study, but sampling is an invasive procedure. Cultured skin fibroblasts seem to improve the biochemical diagnosis, but their growth requires a certain period of time. Our aim was to set up a minimally invasive, fast and reliable analytical procedure to measure CoQ(10) in lymphocytes, to prevent any delay in diagnosing primary CoQ(10) deficiency. HPLC-MS/MS analysis of CoQ(10) showed high sensitivity and specificity. The reference range was established in apparently healthy volunteers (n=33); the mean of CoQ(10) in lymphocytes was 107nmol/g protein (95% confidence interval: 105-120) and 2.0nmol/UCS (95% confidence interval: 2.06-2.46). Therefore, the range was narrower when normalized to units of citrate synthase (UCS) than when normalized to grams of protein. The method was linear from 0.01 to 1µM with a good precision and sensitivity (limit of quantification 0.01µM). Intra-assay and inter-assay coefficients of variation were lower than 13%. Recovery was higher than 95%. In our hands, lymphocytes seem to be a reliable matrix as they reflect intracellular content of CoQ(10). In addition, they can be obtained by a minimally invasive procedure (venipuncture).
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Cromatografia Líquida de Alta Pressão/métodos , Linfócitos/enzimologia , Espectrometria de Massas em Tandem/métodos , Ubiquinona/análogos & derivados , Adolescente , Adulto , Células Cultivadas , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estatísticas não Paramétricas , Ubiquinona/sangueAssuntos
Complexos de ATP Sintetase/genética , Acidose/genética , Cardiomegalia/genética , Retardo do Crescimento Fetal/genética , Glutaratos/metabolismo , Deficiência Intelectual/genética , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Complexos de ATP Sintetase/deficiência , Acidose/metabolismo , Acidose/patologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Criança , Consanguinidade , Análise Mutacional de DNA , Feminino , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , ATPase Trocadora de Hidrogênio-Potássio/deficiência , ATPase Trocadora de Hidrogênio-Potássio/genética , Heterozigoto , Humanos , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Proteínas de Membrana/deficiência , Proteínas Mitocondriais/deficiência , Proteínas/genética , Proteínas/metabolismo , Deleção de Sequência , Proteína Inibidora de ATPaseRESUMO
Neonatal screening of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is of major importance due to the significant morbidity and mortality in undiagnosed patients. MCADD screening has been performed routinely in Galicia since July 2000, and until now 199,943 newborns have been screened. We identified 11 cases of MCADD, which gives an incidence of 1/18,134. During this period, no false negative screens have been detected. At diagnosis, all identified newborns were asymptomatic. Our data showed that octanoylcarnitine (C8) and C8/C10 ratio are the best markers for screening of MCADD. C8 was increased in all patients and C8/C10 was increased in all but one patient.The common mutation, c.985A > G, was found in homozygosity in seven newborns and in compound heterozygosity in three, while one patient did not carry the common mutation at all. In addition, two novel mutations c.245G > C (p.W82S) and c.542A > G (p.D181G) were identified. Ten of the 11 identified newborns did not experience any episodes of decompensation. The patient with the highest level of medium chain acylcarnitines at diagnosis, who was homozygous for the c.985A > G mutation, died at the age of 2 years due to a severe infection.This is the first report of the results from neonatal screening for MCADD in Spain. Our data provide further evidence of the benefits of MCADD screening and contribute to better understanding of this disease.
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Methylenetetrahydrofolate reductase (MTHFR) plays a major role in folate metabolism. Disturbed function of the enzyme results in hyperhomocysteinemia and causes severe vascular and neurological disorders and developmental delay. Five patients suspected of having non-classical homocystinuria due to MTHFR deficiency were examined with respect to their symptoms, MTHFR enzyme activity and genotypes of the MTHFR gene. All patients presented symptoms of severe central nervous system disease. Two patients died, at the ages of 15 months and 14 years. One patient is currently 32 years old, and is being treated with betaine and folinic acid. The other two patients, with an early diagnosis and a severe course of the disease, are currently improving under treatment. MTHFR enzyme activity in the fibroblasts of four of the patients was practically undetectable. We found four novel mutations, three of which were missense changes c.664G> T (p.V218L), c.1316T> C (p.F435S) and c.1733T> G (p.V574G), and the fourth was the 1-bp deletion c.1780delC (p.L590CfsX72). We also found the previously reported nonsense mutation c.1420G> T (p.E470X). All the patients were homozygous. Molecular modelling of the double mutant allele (p.V218L; p.A222V) revealed that affinity for FAD was not affected in this mutant. For the p.E470X mutation, the evidence pointed to nonsense-mediated mRNA decay. In general, genotype-phenotype analysis predicts milder outcomes for patients with missense changes than for those in which mutations led to severe alterations of the MTHFR protein.
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Homocistinúria/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/deficiência , Adolescente , Adulto , Betaína/uso terapêutico , Pré-Escolar , Evolução Fatal , Feminino , Homocistinúria/tratamento farmacológico , Homocistinúria/enzimologia , Humanos , Lactente , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Modelos Moleculares , Tetra-Hidrofolatos/uso terapêutico , TermodinâmicaRESUMO
We screened for PDHA1 mutations in 40 patients with biochemically demonstrated PDHc deficiency or strong clinical suspicion, and found changes with probable pathological significance in 20. Five patients presented new mutations: p.A169V, c.932_938del, c.1143_1144 ins24, c.1146_1159dup and c.510-30G> A, this latter is a new undescribed cause of exon 6 skipping. Another four mutations have been found, and previously reported, in our patients: p.H113D, p.P172L, p.Y243del and p.Y369Q. Eleven patients presented seven known mutations: p.R127Q, p.I166I, p.A198T, p.R263G, p.R302C, p.R378C and c.1142_1145dup. The latter three were found in more than one unrelated patient: p.R302C was detected in a heterozygous girl and a mosaic male, p.R378C in two males and finally, c.1142_1145dup in three females; only one in 20 mothers was found to be a carrier (p.R263G). Apart from those 20 patients, the only alteration detected in one girl with clear PDHc and PDH-E1 deficiency was the silent change c.396A> C (p.R132R), and other eight PDHc deficient patients carry combinations of known infrequent polymorphisms that are overrepresented among our 20 unsolved patients. The importance of these changes on PDH activity is unclear. Investigations in the other PDHc genes are in course in order to elucidate the genetic defect in the unresolved patients.
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Piruvato Desidrogenase (Lipoamida)/genética , Doença da Deficiência do Complexo de Piruvato Desidrogenase/enzimologia , Doença da Deficiência do Complexo de Piruvato Desidrogenase/genética , Western Blotting , Estudos de Casos e Controles , Análise Mutacional de DNA , Feminino , Haplótipos/genética , Humanos , Masculino , Mutação/genética , Seleção de Pacientes , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Creatine transporter (CRTR) deficiency is one of the most frequent causes of X-linked mental retardation. The lack of an effective treatment for this disease, in contrast to creatine (Cr) biosynthesis disorders that respond to Cr monohydrate (CM), led us to analyze the efficacy of a lipophilic molecule derived from Cr, creatine ethyl ester (CEE), in fibroblasts and patients with CRTR deficiency. CM and CEE uptake studies were performed in six controls and four fibroblast cell lines from patients. We found a significant increase in Cr uptake after 72 h of incubation with CEE (500 micromol/L) in patients and control fibroblasts compared to incubation with CM. Subsequently, we assayed the clinical effect of CEE administration in four patients with CRTR deficiency. After 1 year of treatment, a lack of significant improvement in neuropsychological assessment or changes in Cr level in brain (1)H MRS was observed, and CEE was discontinued. In conclusion, this 12-month trial with CEE did not increase the brain concentration of Cr. Our in vitro data lend support to the idea of a certain passive transport of CEE in both pathological and control cells, although more lipophilic molecules or other cell systems that mimic the BBB should be used for a better approach to the in vivo system.
Assuntos
Creatina/análogos & derivados , Fibroblastos/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Deficiência Intelectual Ligada ao Cromossomo X/tratamento farmacológico , Encéfalo/metabolismo , Linhagem Celular , Creatina/administração & dosagem , Creatina/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Resultado do TratamentoRESUMO
Most cases of pyruvate dehydrogenase complex (PDHc) deficiency are attributable to mutations in the PDHA1 gene which encodes the E(1)α subunit, with few cases of mutations in the genes for E(3), E3BP (E(3) binding protein), E(2) and E(1)-phosphatase being reported. Only seven patients with deficiency of the E(1)ß subunit have been described, with mutations in the PDHB gene in six of them. Clinically they presented with a non-specific encephalomyopathy. We report two patients with new mutations in PDHB and Leigh syndrome. Patient 1 was a boy with neonatal onset of hyperlactataemia, corpus callosum hypoplasia and a convulsive encephalopathy. After neurological deterioration, he died at age 5 months. Autopsy revealed the characteristic features of Leigh syndrome. Patient 2, also a boy, presented a milder clinical course. First symptoms were noticed at age 16 months with muscular hypotonia, lactic acidosis and recurrent episodes of somnolence and transient tetraparesis. MRI revealed bilateral signal hyperintensities in the globus pallidus, midbrain and crura cerebri. PDHc and E(1) activities were deficient in fibroblasts in patient 1; in patient 2 PDHc deficiency was found in skeletal muscle. Mutations in PDHA1 were excluded. Sequencing of PDHB revealed a homozygous point mutation (c.302T>C), causing a predicted amino acid change (p.M101T) in patient 1. Patient 2 is compound heterozygote for mutations c.301A>G (p.M101V) and c.313G>A (p.R105Q). All three mutations appear to destabilize the E(1) enzyme with a decrease of both E(1)α and E(1)ß subunits in immunoblot analysis. To our knowledge, these patients with novel PDHB mutations are the first reported with Leigh syndrome.