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1.
Front Immunol ; 10: 2406, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31695692

RESUMO

Severe combined immunodeficiency (SCID), the most severe form of T-cell immunodeficiency, can be screened at birth by quantifying T-cell receptor excision circles (TRECs) in dried blood spot (DBS) samples. Early detection of this condition speeds up the establishment of appropriate treatment and increases the patient's life expectancy. Newborn screening for SCID started in January 2017 in Catalonia, the first Spanish and European region to universally include this testing. The results obtained in the first 2 years of experience are evaluated here. All babies born between January 2017 and December 2018 were screened. TREC quantification in DBS (1.5 mm diameter) was performed with the Enlite Neonatal TREC kit from PerkinElmer (Turku, Finland). In 2018, the retest cutoff in the detection algorithm was updated based on the experience gained in the first year, and changed from 34 to 24 copies/µL. This decreased the retest rate from 3.34 to 1.4% (global retest rate, 2.4%), with a requested second sample rate of 0.23% and a positive detection rate of 0.02%. Lymphocyte phenotype (T, B, NK populations), expression of CD45RA/RO isoforms, percentage and intensity of TCR αß and TCR γδ, presence of HLA-DR+ T lymphocytes, and in vitro lymphocyte proliferation were studied in all patients by flow cytometry. Of 130,903 newborns screened, 30 tested positive, 15 of which were male. During the study period, one patient was diagnosed with SCID: incidence, 1 in 130,903 births in Catalonia. Thirteen patients had clinically significant T-cell lymphopenia (non-SCID) with an incidence of 1 in 10,069 newborns (43% of positive detections). Nine patients were considered false-positive cases because of an initially normal lymphocyte count with normalization of TRECs between 3 and 6 months of life, four infants had transient lymphopenia due to an initially low lymphocyte count with recovery in the following months, and three patients are still under study. The results obtained provide further evidence of the benefits of including this disease in newborn screening programs. Longer follow-up is needed to define the exact incidence of SCID in Catalonia.

2.
Hum Mutat ; 40(10): 1700-1712, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31058414

RESUMO

3-Methylglutaconic aciduria (3-MGA-uria) syndromes comprise a heterogeneous group of diseases associated with mitochondrial membrane defects. Whole-exome sequencing identified compound heterozygous mutations in TIMM50 (c.[341 G>A];[805 G>A]) in a boy with West syndrome, optic atrophy, neutropenia, cardiomyopathy, Leigh syndrome, and persistent 3-MGA-uria. A comprehensive analysis of the mitochondrial function was performed in fibroblasts of the patient to elucidate the molecular basis of the disease. TIMM50 protein was severely reduced in the patient fibroblasts, regardless of the normal mRNA levels, suggesting that the mutated residues might be important for TIMM50 protein stability. Severe morphological defects and ultrastructural abnormalities with aberrant mitochondrial cristae organization in muscle and fibroblasts were found. The levels of fully assembled OXPHOS complexes and supercomplexes were strongly reduced in fibroblasts from this patient. High-resolution respirometry demonstrated a significant reduction of the maximum respiratory capacity. A TIMM50-deficient HEK293T cell line that we generated using CRISPR/Cas9 mimicked the respiratory defect observed in the patient fibroblasts; notably, this defect was rescued by transfection with a plasmid encoding the TIMM50 wild-type protein. In summary, we demonstrated that TIMM50 deficiency causes a severe mitochondrial dysfunction by targeting key aspects of mitochondrial physiology, such as the maintenance of proper mitochondrial morphology, OXPHOS assembly, and mitochondrial respiratory capacity.

4.
Mol Neurobiol ; 55(9): 7216-7228, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29396649

RESUMO

Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and γ-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and α-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system.


Assuntos
Córtex Cerebral/metabolismo , Ácidos Graxos/metabolismo , Grelina/farmacologia , Ácido gama-Aminobutírico/metabolismo , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Citratos/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Deleção de Genes , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Ácidos Cetoglutáricos/metabolismo , Metaboloma/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oxirredução
5.
Pediatrics ; 138(5)2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27940755

RESUMO

We report the case of a 10-year-old Spanish girl with mutations in NADK2 Prenatal central nervous system abnormalities showed ventriculomegaly, colpocephaly, and hypoplasia of the corpus callosum. At birth, axial hypotonia, uncoordinated movements, microcephaly, and generalized cerebellar atrophy were detected. Metabolic investigations revealed high lysine, lactate, and pipecolic acid levels in blood and cerebrospinal fluid. Pyruvate carboxylase and pyruvate dehydrogenase activity in fibroblasts were normal. Beginning at birth she received biotin, thiamine, and carnitine supplementation. A lysine-restricted diet was started when she was 1 month old. Because pipecolic acid was high, pyridoxine was added to treatment. At 3 years old, astatic myoclonic epilepsy appeared, with no response to levetiracetam. We switched pyridoxine to pyridoxal phosphate, with electroclinical improvement. Because the activity of mitochondrial respiratory chain complexes III and IV was slightly low in muscle, other cofactors such as ubidecarenone, idebenone, vitamin E, and creatine were added to the treatment. At 8 years old, plasma acylcarnitine testing was performed, and high levels of 2-trans, 4-cis-decadienoylcarnitine were found. Whole exome sequencing identified a homozygous splice site mutation in NADK2 (c.956+6T>C; p.Trp319Cysfs*21). This substitution generates exon skipping, leading to a truncated protein. In fact, NADK2 messenger RNA and the corresponding protein were almost absent. Now, at 10 years of age she presents with ataxia and incoordination. She has oromotor dysphasia but is able to understand fluid language and is a very friendly girl. We hypothesize that the patient's clinical improvement could be due to her lysine-restricted diet together with cofactors and pyridoxal phosphate administration.


Assuntos
Dieta , Hiperlisinemias/genética , Lisina/administração & dosagem , Proteínas Mitocondriais/genética , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfato de Piridoxal/uso terapêutico , Complexo Vitamínico B/uso terapêutico , Criança , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/terapia , Feminino , Homozigoto , Humanos , Ácido Láctico/sangue , Ácido Láctico/líquido cefalorraquidiano , Lisina/sangue , Lisina/líquido cefalorraquidiano , Doenças Mitocondriais/genética , Malformações do Sistema Nervoso/genética , Ácidos Pipecólicos/sangue , Ácidos Pipecólicos/líquido cefalorraquidiano , RNA Mensageiro/metabolismo
6.
Nefrología (Madr.) ; 36(6): 616-630, nov.-dic. 2016. ilus, tab
Artigo em Espanhol | IBECS | ID: ibc-158751

RESUMO

Introducción: El aumento de la supervivencia de los pacientes con cistinosis y la propia complejidad de la enfermedad explican la necesidad de implementar un proceso de transición guiada desde la medicina pediátrica hasta la del adulto, que permita garantizar el continuumasistencial y posibilite el empoderamiento del paciente desde el cuidado tutelado al autocuidado. Métodos: Revisión bibliográfica, opinión de expertos, encuestas anónimas a pacientes, familiares y asociaciones. Resultados: Elaboración de un documento de transición coordinada, con propuestas concretas por especialidades y de mejora de la adherencia terapéutica y del autocuidado del paciente. El nefrólogo desempeña un papel clave en la transición en la cistinosis debido a la afectación renal que domina la patología y porque la mayoría de los pacientes han recibido un trasplante renal antes de la edad adulta. Conclusión: Se presenta un documento que establece unas recomendaciones y un cronograma para guiar la transición de los adolescentes y adultos jóvenes con cistinosis en nuestro ámbito (AU)


Introduction: Improved outcome and longer life-expectancy in patients with cystinosis, and disease complexity itself, justify planning a guided-transition of affected patients from Pediatrics to adult medicine. The aims of the process are to guarantee the continuum of care and patient empowerment, moving from guardian-care to self-care. Methods: review of articles, expert opinion and anonymous surveys of patients, relatives and patient advocacy groups. Results: elaboration a new document to support and coordinate the transition of patients with cystinosis providing specific proposals in a variety of medical fields, and adherence promotion. Nephrologists play a key role in transition due the fact that most cystinotic patients suffer severe chronic kidney disease, and need kidney transplantation before adulthood. Conclusion: we present a document providing recommendations and suggesting a chronogram to help the process of transition of adolescents and young adults with cystinosis in our area (AU)


Assuntos
Humanos , Adolescente , Adulto Jovem , Cistinose/terapia , Planejamento de Assistência ao Paciente/organização & administração , Cuidado Transicional/organização & administração , Padrões de Prática Médica , Insuficiência Renal Crônica/prevenção & controle , Doenças Raras/epidemiologia , Perfil de Impacto da Doença
7.
PLoS One ; 11(9): e0163762, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27689355

RESUMO

INTRODUCTION: Parenteral nutrition (PN) dependence in short bowel syndrome (SBS) patients is linked to the functionality of the remnant small bowel (RSB). Patients may wean off PN following a period of intestinal adaptation that restores this functionality. Currently, plasma citrulline is the standard biomarker for monitoring intestinal functionality and adaptation. However, available studies reveal that the relationship the biomarker with the length and function of the RSB is arguable. Thus, having additional biomarkers would improve pointing out PN weaning. AIM: By measuring concomitant changes in citrulline and the novel biomarker apolipoprotein AIV (Apo AIV), as well as taking into account the anatomy of the RSB, this exploratory study aims to a better understanding of the intestinal adaptation process and characterization of the SBS patients under PN. METHODS: Thirty four adult SBS patients were selected and assigned to adapted (aSBS) and non-adapted (nSBS) groups after reconstructive surgeries. Remaining jejunum and ileum lengths were recorded. The aSBS patients were either on an oral diet (ORAL group), those with intestinal insufficiency, or on oral and home parenteral nutrition (HPN group), those with chronic intestinal failure. Apo AIV and citrulline were analyzed in plasma samples after overnight fasting. An exploratory ROC analysis using citrulline as gold standard was performed. RESULTS: Biomarkers, Apo AIV and citrulline showed a significant correlation with RSBL in aSBS patients. In jejuno-ileocolic patients, only Apo AIV correlated with RSBL (rb = 0.54) and with ileum length (rb = 0.84). In patients without ileum neither biomarker showed any correlation with RSBL. ROC analysis indicated the Apo AIV cut-off value to be 4.6 mg /100 mL for differentiating between the aSBS HPN and ORAL groups. CONCLUSIONS: Therefore, in addition to citrulline, Apo AIV can be set as a biomarker to monitor intestinal adaptation in SBS patients. As short bowel anatomy is shown to influence citrulline and Apo AIV plasma values, both biomarkers complement each other furnishing a new insight to manage PN dependence.

8.
Nefrologia ; 36(6): 616-630, 2016.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-27595514

RESUMO

INTRODUCTION: Improved outcome and longer life-expectancy in patients with cystinosis, and disease complexity itself, justify planning a guided-transition of affected patients from Pediatrics to adult medicine. The aims of the process are to guarantee the continuum of care and patient empowerment, moving from guardian-care to self-care. METHODS: review of articles, expert opinion and anonymous surveys of patients, relatives and patient advocacy groups. RESULTS: elaboration a new document to support and coordinate the transition of patients with cystinosis providing specific proposals in a variety of medical fields, and adherence promotion. Nephrologists play a key role in transition due the fact that most cystinotic patients suffer severe chronic kidney disease, and need kidney transplantation before adulthood. CONCLUSION: we present a document providing recommendations and suggesting a chronogram to help the process of transition of adolescents and young adults with cystinosis in our area.


Assuntos
Cistinose/terapia , Transição para Assistência do Adulto , Adolescente , Adulto , Criança , Humanos , Transplante de Rim , Pediatria , Insuficiência Renal Crônica/terapia , Autocuidado , Adulto Jovem
9.
Mitochondrion ; 30: 51-8, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27374853

RESUMO

We evaluated the coenzyme Q10 (CoQ) levels in patients who were diagnosed with mitochondrial oxidative phosphorylation (OXPHOS) and non-OXPHOS disorders (n=72). Data from the 72 cases in this study revealed that 44.4% of patients showed low CoQ concentrations in either their skeletal muscle or skin fibroblasts. Our findings suggest that secondary CoQ deficiency is a common finding in OXPHOS and non-OXPHOS disorders. We hypothesize that cases of CoQ deficiency associated with OXPHOS defects could be an adaptive mechanism to maintain a balanced OXPHOS, although the mechanisms explaining these deficiencies and the pathophysiological role of secondary CoQ deficiency deserves further investigation.


Assuntos
Doenças Mitocondriais/patologia , Fosforilação Oxidativa , Ubiquinona/análogos & derivados , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Músculos/patologia , Prevalência , Pele/patologia , Ubiquinona/deficiência , Adulto Jovem
10.
Mitochondrion ; 26: 72-80, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26688339

RESUMO

Mutations in NFU1 were recently identified in patients with fatal encephalopathy. NFU1 is an iron-sulfur cluster protein necessary for the activity of the mitochondrial respiratory chain complexes I-II and the synthesis of lipoic acid. We report two NFU1 compound heterozygous individuals with normal complex I and lipoic acid-dependent enzymatic activities and low, but detectable, levels of lipoylated proteins. We demonstrated a leaky splicing regulation due to a splice site mutation (c.545+5G>A) that produces small amounts of wild type NFU1 mRNA that might result in enough protein to partially lipoylate and restore the activity of lipoic acid-dependent enzymes and the assembly and activity of complex I. These results allowed us to gain insights into the molecular basis underlying this disease and should be considered for the diagnosis of NFU1 patients.


Assuntos
Encefalopatias Metabólicas/diagnóstico por imagem , Encefalopatias Metabólicas/genética , Proteínas de Transporte/genética , Mutação , Sítios de Splice de RNA , Processamento de RNA , Encefalopatias Metabólicas/metabolismo , Proteínas de Transporte/metabolismo , Feminino , Humanos , Lactente , Lipoilação/genética , Masculino , Radiografia
11.
Brain ; 139(Pt 1): 31-8, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26657515

RESUMO

Thiamine transporter-2 deficiency is caused by mutations in the SLC19A3 gene. As opposed to other causes of Leigh syndrome, early administration of thiamine and biotin has a dramatic and immediate clinical effect. New biochemical markers are needed to aid in early diagnosis and timely therapeutic intervention. Thiamine derivatives were analysed by high performance liquid chromatography in 106 whole blood and 38 cerebrospinal fluid samples from paediatric controls, 16 cerebrospinal fluid samples from patients with Leigh syndrome, six of whom harboured mutations in the SLC19A3 gene, and 49 patients with other neurological disorders. Free-thiamine was remarkably reduced in the cerebrospinal fluid of five SLC19A3 patients before treatment. In contrast, free-thiamine was slightly decreased in 15.2% of patients with other neurological conditions, and above the reference range in one SLC19A3 patient on thiamine supplementation. We also observed a severe deficiency of free-thiamine and low levels of thiamine diphosphate in fibroblasts from SLC19A3 patients. Surprisingly, pyruvate dehydrogenase activity and mitochondrial substrate oxidation rates were within the control range. Thiamine derivatives normalized after the addition of thiamine to the culture medium. In conclusion, we found a profound deficiency of free-thiamine in the CSF and fibroblasts of patients with thiamine transporter-2 deficiency. Thiamine supplementation led to clinical improvement in patients early treated and restored thiamine values in fibroblasts and cerebrospinal fluid.


Assuntos
Doença de Leigh/dietoterapia , Doença de Leigh/metabolismo , Proteínas de Membrana Transportadoras/deficiência , Tiamina/metabolismo , Tiamina/uso terapêutico , Adolescente , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Recém-Nascido , Doença de Leigh/sangue , Doença de Leigh/líquido cefalorraquidiano , Doença de Leigh/genética , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Complexo Piruvato Desidrogenase/metabolismo , Tiamina/sangue , Tiamina/líquido cefalorraquidiano , Tiamina Pirofosfato/metabolismo
12.
Nefrologia ; 35(3): 304-21, 2015.
Artigo em Inglês, Espanhol | MEDLINE | ID: mdl-26523297

RESUMO

INTRODUCTION: Cystinosis is a rare lysosomal systemic disease that mainly affects the kidney and the eye. Patients with cystinosis begin renal replacement therapy during the first decade of life in absence of treatment. Prognosis of cystinosis depends on early diagnosis, and prompt starting and good compliance with cysteamine treatment. Kidney disease progression, extra-renal complications and shorter life expectancy are more pronounced in those patients that do not follow treatment. The objective of this work was to elaborate recommendations for the comprehensive care of cystinosis and the facilitation of patient transition from paediatric to adult treatment, based on clinical experience. The goal is to reduce the impact of the disease, and to improve patient quality of life and prognosis. METHODS: Bibliographic research and consensus meetings among a multidisciplinary professional team of experts in the clinical practice, with cystinotic patients (T-CiS.bcn group) from 5 hospitals located in Barcelona. RESULTS: This document gathers specific recommendations for diagnosis, treatment and multidisciplinary follow-up of cystinotic patients in the following areas: nephrology, dialysis,renal transplant, ophthalmology, endocrinology, neurology, laboratory, genetic counselling,nursing and pharmacy. CONCLUSIONS: A reference document for the comprehensive care of cystinosis represents a support tool for health professionals who take care of these patients. It is based on the following main pillars: (a) a multi-disciplinary approach, (b) appropriate disease monitoring and control of intracellular cystine levels in leukocytes, (c) the importance of adherence to treatment with cysteamine, and (d) the promotion of patient self-care by means of disease education programmes. All these recommendations will lead us, in a second phase, to create a coordinated transition model between paediatric and adult care services which will contemplate the specific needs of cystinosis.


Assuntos
Assistência Integral à Saúde/normas , Cistinose/terapia , Transição para Assistência do Adulto/normas , Adolescente , Adulto , Sistemas de Transporte de Aminoácidos Neutros/deficiência , Sistemas de Transporte de Aminoácidos Neutros/genética , Doenças da Córnea/diagnóstico , Doenças da Córnea/etiologia , Doenças da Córnea/terapia , Cisteamina/uso terapêutico , Cistinose/complicações , Cistinose/diagnóstico , Cistinose/genética , Gerenciamento Clínico , Diagnóstico Precoce , Doenças do Sistema Endócrino/diagnóstico , Doenças do Sistema Endócrino/etiologia , Doenças do Sistema Endócrino/terapia , Aconselhamento Genético , Humanos , Comunicação Interdisciplinar , Falência Renal Crônica/etiologia , Falência Renal Crônica/prevenção & controle , Falência Renal Crônica/terapia , Transplante de Rim , Doenças do Sistema Nervoso/diagnóstico , Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/terapia , Educação de Pacientes como Assunto , Qualidade de Vida , Autocuidado
13.
J Lipid Res ; 56(10): 1926-35, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26239048

RESUMO

Niemann-Pick type C (NPC) is a progressive neurodegenerative disease characterized by lysosomal/endosomal accumulation of unesterified cholesterol and glycolipids. Recent studies have shown that plasma cholestane-3ß,5α,6ß-triol (CT) and 7-ketocholesterol (7-KC) could be potential biomarkers for the diagnosis of NPC patients. We aimed to know the sensitivity and specificity of these biomarkers for the diagnosis of NPC compared with other diseases that can potentially lead to oxysterol alterations. We studied 107 controls and 122 patients including 16 with NPC, 3 with lysosomal acid lipase (LAL) deficiency, 8 with other lysosomal diseases, 5 with galactosemia, 11 with cerebrotendinous xanthomatosis (CTX), 3 with Smith-Lemli-Opitz, 14 with peroxisomal biogenesis disorders, 19 with unspecific hepatic diseases, 13 with familial hypercholesterolemia, and 30 with neurological involvement and no evidence of an inherited metabolic disease. CT and 7-KC were analyzed by HPLC-ESI-MS/MS as mono-dimethylglycine derivatives. Levels of 7-KC were high in most of the studied diseases, whereas those of CT were only high in NPC, LAL, and CTX patients. Consequently, although CT is a sensitive biomarker of NPC disease, including those cases with doubtful filipin staining, it is not specific. 7-KC is a very unspecific biomarker.


Assuntos
Colestanóis/sangue , Cetocolesteróis/sangue , Doença de Niemann-Pick Tipo C/sangue , Doença de Wolman/sangue , Xantomatose Cerebrotendinosa/sangue , Adolescente , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas por Ionização por Electrospray/métodos , Doença de Wolman/diagnóstico , Xantomatose Cerebrotendinosa/diagnóstico
14.
Neurotherapeutics ; 12(4): 874-86, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26169295

RESUMO

Aminoglycoside antibiotics, such as gentamicin, may induce premature termination codon (PTC) readthrough and elude the nonsense-mediated mRNA decay mechanism. Because PTCs are frequently involved in lysosomal diseases, readthrough compounds may be useful as potential therapeutic agents. The aim of our study was to identify patients responsive to gentamicin treatment in order to be used as positive controls to further screen for other PTC readthrough compounds. With this aim, fibroblasts from 11 patients affected by 6 different lysosomal diseases carrying PTCs were treated with gentamicin. Treatment response was evaluated by measuring enzymatic activity, abnormal metabolite accumulation, mRNA expression, protein localization, and cell viability. The potential effect of readthrough was also analyzed by in silico predictions. Results showed that fibroblasts from 5/11 patients exhibited an up to 3-fold increase of enzymatic activity after gentamicin treatment. Accordingly, cell lines tested showed enhanced well-localized protein and/or increased mRNA expression levels and/or reduced metabolite accumulation. Interestingly, these cell lines also showed increased enzymatic activity after PTC124 treatment, which is a PTC readthrough-promoting compound. In conclusion, our results provide a proof-of-concept that PTCs can be effectively suppressed by readthrough drugs, with different efficiencies depending on the genetic context. The screening of new compounds with readthrough activity is a strategy that can be used to develop efficient therapies for diseases caused by PTC mutations.


Assuntos
Códon sem Sentido/genética , Fibroblastos/metabolismo , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/patologia , Esfingomielina Fosfodiesterase/genética , Acetilglucosaminidase/genética , Acetilglucosaminidase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Fibroblastos/efeitos dos fármacos , Gentamicinas/farmacologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosaminoglicanos/genética , Glicosaminoglicanos/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Mutação/genética , Oxidiazóis/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo
15.
Nefrología (Madr.) ; 35(3): 304-321, mayo-jun. 2015. ilus, tab
Artigo em Inglês | IBECS | ID: ibc-140060

RESUMO

Introduction: Cystinosis is a rare lysosomal systemic disease that mainly affects the kidney and the eye. Patients with cystinosis begin renal replacement therapy during the first decade of life in absence of treatment. Prognosis of cystinosis depends on early diagnosis, and prompt starting and good compliance with cysteamine treatment. Kidney disease progression, extra-renal complications and shorter life expectancy are more pronounced in those patients that do not follow treatment. The objective of this work was to elaborate recommendations for the comprehensive care of cystinosis and the facilitation of patient transition from paediatric to adult treatment, based on clinical experience. The goal is to reduce the impact of the disease, and to improve patient quality of life and prognosis. Methods: Bibliographic research and consensus meetings among a multidisciplinary professional team of experts in the clinical practice, with cystinotic patients (T-CiS.bcn group) from 5 hospitals located in Barcelona. Results: This document gathers specific recommendations for diagnosis, treatment and multidisciplinary follow-up of cystinotic patients in the following areas: nephrology, dialysis, renal transplant, ophthalmology, endocrinology, neurology, laboratory, genetic counselling, nursing and pharmacy. Conclusions: A reference document for the comprehensive care of cystinosis represents a support tool for health professionals who take care of these patients. It is based on the following main pillars: (a) a multi-disciplinary approach, (b) appropriate disease monitoring and control of intracellular cystine levels in leukocytes, (c) the importance of adherence to treatment with cysteamine, and (d) the promotion of patient self-care by means of disease education programmes. All these recommendations will lead us, in a second phase, to create a coordinated transition model between paediatric and adult care services which will contemplate the specific needs of cystinosis (AU)


Introducción: La cistinosis es una enfermedad lisosomal minoritaria de expresión sistémica con especial afectación renal y oftalmológica, en la que los pacientes inician terapia renal sustitutiva en la primera década de la vida en ausencia de tratamiento. El pronóstico de la cistinosis depende del diagnóstico precoz, la pronta instauración del tratamiento con cisteamina y el buen cumplimiento terapéutico. La progresión de la enfermedad renal y de las complicaciones extrarrenales y una menor supervivencia, son más acentuadas en pacientes no adherentes. Objetivo: El objetivo de este trabajo fue la elaboración de unas recomendaciones para la atención integral de la cistinosis y la transición del adolescente a la medicina del adulto, basadas en la experiencia clínica, con el fin de reducir el impacto de la enfermedad y mejorar la calidad de vida y el pronóstico del paciente. Método: Búsqueda bibliográfica y reuniones de consenso de un equipo multidisciplinar de expertos en la práctica clínica con pacientes afectos de cistinosis (Grupo T-CiS.bcn), procedentes de 5 hospitales localizados en Barcelona. Resultados: El documento recoge recomendaciones específicas y necesarias para el diagnóstico, tratamiento y seguimiento multidisciplinar de la cistinosis en las siguientes áreas: nefrología, diálisis, trasplante renal, oftalmología, endocrinología, neurología, laboratorio, consejo genético, enfermería y farmacia. Conclusiones: Disponer de un documento de referencia para la atención integral de la cistinosis constituye una herramienta de soporte para los profesionales de la salud que asisten a estos pacientes. Los principales pilares en los que se sustenta son: a) el enfoque multidisciplinar, b) la adecuada monitorización de la enfermedad y control de los niveles de cistina intraleucocitarios, c) la importancia de la adherencia al tratamiento con cisteamina y d) la promoción del autocuidado del paciente mediante programas de educación en la enfermedad. Todo ello conducirá, en una segunda fase, a la elaboración de un modelo de transición coordinado entre los servicios de pediatría y de adultos que contemple las necesidades específicas de la cistinosis (AU)


Assuntos
Adolescente , Adulto , Feminino , Humanos , Masculino , Cistinose/epidemiologia , Cistinose/prevenção & controle , /métodos , /tendências , Cisteamina/administração & dosagem , Cisteamina/análise , Prognóstico , Diagnóstico Precoce , Síndrome de Fanconi/complicações , Síndrome de Fanconi/diagnóstico , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/etiologia , Diálise Renal/métodos , Diálise Renal , Biologia Molecular/métodos
16.
PLoS One ; 9(5): e98109, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24858472

RESUMO

FATP1 mediates skeletal muscle cell fatty acid import, yet its intracellular localization and metabolic control role are not completely defined. Here, we examine FATP1 localization and metabolic effects of its overexpression in mouse skeletal muscle. The FATP1 protein was detected in mitochondrial and plasma membrane fractions, obtained by differential centrifugation, of mouse gastrocnemius muscle. FATP1 was most abundant in purified mitochondria, and in the outer membrane and soluble intermembrane, but not in the inner membrane plus matrix, enriched subfractions of purified mitochondria. Immunogold electron microscopy localized FATP1-GFP in mitochondria of transfected C2C12 myotubes. FATP1 was overexpressed in gastrocnemius mouse muscle, by adenovirus-mediated delivery of the gene into hindlimb muscles of newborn mice, fed after weaning a chow or high-fat diet. Compared to GFP delivery, FATP1 did not alter body weight, serum fed glucose, insulin and triglyceride levels, and whole-body glucose tolerance, in either diet. However, fatty acid levels were lower and ß-hydroxybutyrate levels were higher in FATP1- than GFP-mice, irrespective of diet. Moreover, intramuscular triglyceride content was lower in FATP1- versus GFP-mice regardless of diet, and ß-hydroxybutyrate content was unchanged in high-fat-fed mice. Electroporation-mediated FATP1 overexpression enhanced palmitate oxidation to CO2, but not to acid-soluble intermediate metabolites, while CO2 production from ß-hydroxybutyrate was inhibited and that from glucose unchanged, in isolated mouse gastrocnemius strips. In summary, FATP1 was localized in mitochondria, in the outer membrane and intermembrane parts, of mouse skeletal muscle, what may be crucial for its metabolic effects. Overexpressed FATP1 enhanced disposal of both systemic fatty acids and intramuscular triglycerides. Consistently, it did not contribute to the high-fat diet-induced metabolic dysregulation. However, FATP1 lead to hyperketonemia, likely secondary to the sparing of ketone body oxidation by the enhanced oxidation of fatty acids.


Assuntos
Proteínas de Transporte de Ácido Graxo/metabolismo , Corpos Cetônicos/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Ácido 3-Hidroxibutírico/metabolismo , Adenoviridae/genética , Animais , Glicemia/metabolismo , Linhagem Celular , Coenzima A-Transferases/genética , Dieta Hiperlipídica/efeitos adversos , Proteínas de Transporte de Ácido Graxo/genética , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hidroximetilglutaril-CoA Sintase/genética , Insulina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Células Musculares/citologia , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Esquelético/efeitos dos fármacos , Oxirredução , Palmitatos/metabolismo , Proteínas Quinases/genética , Transporte Proteico/efeitos dos fármacos , Triglicerídeos/metabolismo
17.
J Inherit Metab Dis ; 37(1): 53-62, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23774949

RESUMO

Primary coenzyme Q10 (CoQ10) deficiencies are associated with mutations in genes encoding enzymes important for its biosynthesis and patients are responsive to CoQ10 supplementation. Early treatment allows better prognosis of the disease and therefore, early diagnosis is desirable. The complex phenotype and genotype and the frequent secondary CoQ10 deficiencies make it difficult to achieve a definitive diagnosis by direct quantification of CoQ10. We developed a non-radioactive methodology for the quantification of CoQ10 biosynthesis in fibroblasts that allows the identification of primary deficiencies. Fibroblasts were incubated 72 h with 28 µmol/L (2)H3-mevalonate or 1.65 mmol/L (13)C6-p-hydroxybenzoate. The newly synthesized (2)H3- and (13)C6- labelled CoQ10 were analysed by high performance liquid chromatography-tandem mass spectrometry. The mean and the reference range for (13)C6-CoQ10 and (2)H3-CoQ10 biosynthesis were 0.97 (0.83-1.1) and 0.13 (0.09-0.17) nmol/Unit of citrate synthase, respectively. We validated the methodology through the study of one patient with COQ2 mutations and six patients with CoQ10 deficiency secondary to other inborn errors of metabolism. Afterwards we investigated 16 patients' fibroblasts and nine showed decreased CoQ10 biosynthesis. Therefore, the next step is to study the COQ genes in order to reach a definitive diagnosis in these nine patients. In the patients with normal rates the deficiency is probably secondary. In conclusion, we have developed a non-invasive non-radioactive method suitable for the detection of defects in CoQ10 biosynthesis, which offers a good tool for the stratification of patients with these treatable mitochondrial diseases.


Assuntos
Ataxia/diagnóstico , Ataxia/metabolismo , Fibroblastos/metabolismo , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/metabolismo , Debilidade Muscular/diagnóstico , Debilidade Muscular/metabolismo , Mutação , Ubiquinona/análogos & derivados , Ubiquinona/deficiência , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Citrato (si)-Sintase/metabolismo , Genótipo , Humanos , Técnicas de Diagnóstico Molecular , Fenótipo , Valores de Referência , Reprodutibilidade dos Testes , Pele/metabolismo , Espectrometria de Massas em Tandem , Fatores de Tempo , Ubiquinona/biossíntese , Ubiquinona/metabolismo
18.
Hum Mol Genet ; 23(7): 1907-15, 2014 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-24256811

RESUMO

Cofactor disorders of mitochondrial energy metabolism are a heterogeneous group of diseases with a wide variety of clinical symptoms, particular metabolic profiles and variable enzymatic defects. Mutations in NFU1, BOLA3, LIAS and IBA57 have been identified in patients with deficient lipoic acid-dependent enzymatic activities and defects in the assembly and activity of the mitochondrial respiratory chain complexes. Here, we report a patient with an early onset fatal lactic acidosis presenting a biochemical phenotype compatible with a combined defect of pyruvate dehydrogenase (PDHC) and 2-ketoglutarate dehydrogenase (2-KGDH) activities, which suggested a deficiency in lipoic acid metabolism. Immunostaining analysis showed that lipoylated E2-PDH and E2-KGDH were extremely reduced in this patient. However, the absence of glycine elevation, the normal activity of the glycine cleavage system and the normal lipoylation of the H protein suggested a defect of lipoic acid transfer to particular proteins rather than a general impairment of lipoic acid biosynthesis as the potential cause of the disease. By analogy with yeast metabolism, we postulated LIPT1 as the altered candidate gene causing the disease. Sequence analysis of the human LIPT1 identified two heterozygous missense mutations (c.212C>T and c.292C>G), segregating in different alleles. Functional complementation experiments in patient's fibroblasts demonstrated that these mutations are disease-causing and that LIPT1 protein is required for lipoylation and activation of 2-ketoacid dehydrogenases in humans. These findings expand the spectrum of genetic defects associated with lipoic acid metabolism and provide the first evidence of a lipoic acid transfer defect in humans.


Assuntos
Aciltransferases/genética , Lipoilação/genética , Oxo-Ácido-Liases/genética , Acidose Láctica/genética , Acidose Láctica/mortalidade , Erros Inatos do Metabolismo dos Aminoácidos/genética , Animais , Células COS , Células Cultivadas , Metabolismo Energético/genética , Feminino , Humanos , Recém-Nascido , Complexo Cetoglutarato Desidrogenase/deficiência , Complexo Cetoglutarato Desidrogenase/genética , Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Complexo Piruvato Desidrogenase/genética , Ácido Tióctico/metabolismo
19.
J Inherit Metab Dis ; 37(3): 439-46, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24347096

RESUMO

Coenzyme Q10 (CoQ10) plays a key role in the exchange of electrons in lysosomal membrane, which contributes to protons' translocation into the lumen and to the acidification of intra-lysosomal medium, which is essential for the proteolytic function of hydrolases responsible -when deficient- of a wide range of inherited lysosomal diseases such as Sanfilippo syndromes. Our aim was to evaluate whether treatment with CoQ10 or with an antioxidant cocktail (α-tocopherol, N-acetylcysteine and α-lipoic acid) were able to ameliorate the biochemical phenotype in cultured fibroblasts of Sanfilippo patients. Basal CoQ10 was analyzed in fibroblasts and Sanfilippo A patients showed decreased basal levels. However, no dysfunction in the CoQ10 biosynthesis pathways was found, revealing for the first time a secondary CoQ10 deficiency in Sanfilippo A fibroblasts. Cultured fibroblasts from five patients affected by Sanfilippo A and B diseases were treated with CoQ10 and an antioxidant cocktail. Upon CoQ10 treatment, none of the Sanfilippo A fibroblasts increased their residual enzymatic activity, but the two Sanfilippo B cell lines showed a statistically significant increase of their residual activity. The antioxidant treatment had no effect on the residual activity in all tested cell lines. Moreover, one Sanfilippo A and two Sanfilippo B fibroblasts showed a statistically significant reduction of glycosaminoglycans accumulation both, after 50 µmol/L CoQ10 and antioxidant treatment. Fibroblasts responsive to treatment enhanced their exocytosis levels. Our results are encouraging as some cellular alterations observed in Sanfilippo syndrome can be partially restored by CoQ10 or other antioxidant treatment in some patients.


Assuntos
Antioxidantes/uso terapêutico , Fibroblastos/efeitos dos fármacos , Mucopolissacaridose III/tratamento farmacológico , Ubiquinona/análogos & derivados , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Exocitose , Glicosaminoglicanos/metabolismo , Humanos , Lisossomos/metabolismo , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Ubiquinona/uso terapêutico
20.
Orphanet J Rare Dis ; 8: 102, 2013 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-23842438

RESUMO

BACKGROUND: Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common inherited defect in the mitochondrial fatty acid oxidation pathway, resulting in significant morbidity and mortality in undiagnosed patients.Newborn screening (NBS) has considerably improved MCADD outcome, but the risk of complication remains in some patients. The aim of this study was to evaluate the relationship between genotype, biochemical parameters and clinical data at diagnosis and during follow-up, in order to optimize monitoring of these patients. METHODS: We carried out a multicenter study in southwest Europe, of MCADD patients detected by NBS. Evaluated NBS data included free carnitine (C0) and the acylcarnitines C8, C10, C10:1 together with C8/C2 and C8/C10 ratios, clinical presentation parameters and genotype, in 45 patients. Follow-up data included C0 levels, duration of carnitine supplementation and occurrence of metabolic crises. RESULTS: C8/C2 ratio and C8 were the most accurate biomarkers of MCADD in NBS. We found a high number of patients homozygous for the prevalent c.985A > G mutation (75%). Moreover, in these patients C8, C8/C10 and C8/C2 were higher than in patients with other genotypes, while median value of C0 was significantly lower (23 µmol/L vs 36 µmol/L).The average follow-up period was 43 months. To keep carnitine levels within the normal range, carnitine supplementation was required in 82% of patients, and for a longer period in patients homozygotes for the c.985A>G mutation than in patients with other genotypes (average 31 vs 18 months). Even with treatment, median C0 levels remained lower in homozygous patients than in those with other genotypes (14 µmol/L vs 22 µmol/L).Two patients died and another three suffered a metabolic crisis, all of whom were homozygous for the c.985 A>G mutation. CONCLUSIONS: Our data show a direct association between homozygosity for c.985A>G and lower carnitine values at diagnosis, and a higher dose of carnitine supplementation for maintenance within the normal range. This study contributes to a better understanding of the relationship between genotype and phenotype in newborn patients with MCADD detected through screening which could be useful in improving follow-up strategies and clinical outcome.


Assuntos
Acil-CoA Desidrogenase/deficiência , Cardiomiopatias/epidemiologia , Carnitina/sangue , Carnitina/deficiência , Hiperamonemia/epidemiologia , Erros Inatos do Metabolismo Lipídico/diagnóstico , Doenças Musculares/epidemiologia , Triagem Neonatal/métodos , Cardiomiopatias/diagnóstico , Cardiomiopatias/tratamento farmacológico , Carnitina/administração & dosagem , Carnitina/análogos & derivados , Suplementos Nutricionais , Feminino , Estudos de Associação Genética , Genótipo , Homozigoto , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/tratamento farmacológico , Incidência , Recém-Nascido , Erros Inatos do Metabolismo Lipídico/tratamento farmacológico , Erros Inatos do Metabolismo Lipídico/epidemiologia , Masculino , Doenças Musculares/diagnóstico , Doenças Musculares/tratamento farmacológico , Fenótipo , Prevalência , Espanha/epidemiologia , Complexo Vitamínico B/administração & dosagem , Complexo Vitamínico B/sangue
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