RESUMEN
A simple and rapid HPLC-MS/MS analytical method was developed and validated for the determination of methylmalonic acid (MMA) in human serum without a derivatization step. Serum samples (200 µl) were pretreated using a simple method based on ultrafiltration using a VIVASPIN 500 ultrafiltration column. Chromatographic separation was achieved on a Luna Omega C18 column with a PS C18 precolumn guard by gradient elution using 0.1% (v/v) formic acid in water (mobile phase A) and 0.5% (v/v) formic acid in acetonitrile (mobile phase B) at a flow rate of 0.2 ml min-1 . The total run time of the analysis was 4.5 min. Negative electrospray ionization and multiple reaction monitoring mode were used. The lower limit of detection and lower limit of quantification for MMA were determined to be 13.6 and 42.3 nmol L-1 , respectively. The developed method enabled the quantification of MMA in a wide linear range of 42.3-4230 nmol L-1 with a correlation coefficient of 0.9991.
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Ácido Metilmalónico , Espectrometría de Masas en Tándem , Humanos , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Formiatos , Reproducibilidad de los ResultadosRESUMEN
A simple and fast high-performance liquid chromatography with tandem mass spectrometry method for quantification of tazobactam, piperacillin, and meropenem in human plasma has been developed and validated. Simple sample preparation with a volume of 10 µL was done by protein precipitation with a mixture of methanol-acetonitrile-water (6:2:2, v/v/v). Chromatographic separation was achieved on a Luna column with a precolumn security guard by gradient elution using a mobile phase consisting of water with the addition of 0.1% formic acid (component A) and mixture methanol-acetonitrile (8:2, v/v) with the addition of 0.1% formic acid (component B). The run time was 2.7 min. The lower limits of detection and lower limits of quantification were for piperacillin 0.03 and 0.1 mg/L, for meropenem 0.04 and 0.2 mg/L and for tazobactam 0.16 and 0.5 mg/L. The validated method was used for therapeutic monitoring of tazobactam, piperacillin, and meropenem in samples of patients treated in the intensive care unit.
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Antibacterianos/sangre , Monitoreo de Drogas/métodos , Plasma/química , Cromatografía Líquida de Alta Presión/métodos , Humanos , Límite de Detección , Meropenem/sangre , Piperacilina/sangre , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem/métodos , Tazobactam/sangre , Inhibidores de beta-Lactamasas/sangreRESUMEN
Inherited disorders of mitochondrial energy metabolism form a large and heterogeneous group of metabolic diseases. More than 250 gene defects have been reported to date and this number continues to grow. Mitochondrial diseases can be grouped into (1) disorders of oxidative phosphorylation (OXPHOS) subunits and their assembly factors, (2) defects of mitochondrial DNA, RNA and protein synthesis, (3) defects in the substrate-generating upstream reactions of OXPHOS, (4) defects in relevant cofactors and (5) defects in mitochondrial homeostasis. Deficiency of more than one respiratory chain enzyme is a common finding. Combined defects are found in 49 % of the known disease-causing genes of mitochondrial energy metabolism and in 57 % of patients with OXPHOS defects identified in our diagnostic centre. Combined defects of complexes I, III, IV and V are typically due to deficiency of mitochondrial DNA replication, RNA metabolism or translation. Defects in cofactors can result in combined defects of various combinations, and defects of mitochondrial homeostasis can result in a generalised decrease of all OXPHOS enzymes. Noteworthy, identification of combined defects can be complicated by different degrees of severity of each affected enzyme. Furthermore, even defects of single respiratory chain enzymes can result in combined defects due to aberrant formation of respiratory chain supercomplexes. Combined OXPHOS defects have a great variety of clinical manifestations in terms of onset, course severity and tissue involvement. They can present as classical encephalomyopathy but also with hepatopathy, nephropathy, haematologic findings and Perrault syndrome in a subset of disorders.
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Enfermedades Mitocondriales/genética , Metabolismo Energético/genética , Humanos , Fosforilación OxidativaRESUMEN
Thiamine pyrophosphate (TPP) is an essential cofactor of the cytosolic transketolase and of three mitochondrial enzymes involved in the oxidative decarboxylation of either pyruvate, α-ketoglutarate or branched chain amino acids. Thiamine is taken up by specific transporters into the cell and converted to the active TPP by thiamine pyrophosphokinase (TPK) in the cytosol from where it can be transported into mitochondria. Here, we report five individuals from three families presenting with variable degrees of ataxia, psychomotor retardation, progressive dystonia, and lactic acidosis. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate but normal pyruvate dehydrogenase complex activity in the presence of excess TPP. A reduced concentration of TPP was found in the muscle and blood. Mutation analysis of TPK1 uncovered three missense, one splice-site, and one frameshift mutation resulting in decreased TPK protein levels.
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Anomalías Múltiples/enzimología , Encefalopatías Metabólicas/enzimología , Redes y Vías Metabólicas/genética , Ácido Pirúvico/metabolismo , Tiamina Pirofosfoquinasa/deficiencia , Anomalías Múltiples/tratamiento farmacológico , Anomalías Múltiples/genética , Acidosis Láctica/enzimología , Acidosis Láctica/genética , Adolescente , Secuencia de Aminoácidos , Secuencia de Bases , Encefalopatías Metabólicas/tratamiento farmacológico , Encefalopatías Metabólicas/genética , Niño , Análisis Mutacional de ADN , Pruebas de Enzimas , Resultado Fatal , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Mutación , Oxidación-Reducción , Linaje , Tiamina Pirofosfoquinasa/genética , Tiamina/sangre , Tiamina/metabolismo , Tiamina/uso terapéuticoRESUMEN
Defects of mitochondrial oxidative phosphorylation constitute a clinical and genetic heterogeneous group of disorders affecting multiple organ systems at varying age. Biochemical analysis of biopsy material demonstrates isolated or combined deficiency of mitochondrial respiratory chain enzyme complexes. Co-occurrence of impaired activity of the pyruvate dehydrogenase complex has been rarely reported so far and is not yet fully understood. We investigated two siblings presenting with severe neonatal lactic acidosis, hypotonia, and intractable cardiomyopathy; both died within the first months of life. Muscle biopsy revealed a peculiar biochemical defect consisting of a combined deficiency of respiratory chain complexes I, II, and II+III accompanied by a defect of the pyruvate dehydrogenase complex. Joint exome analysis of both affected siblings uncovered a homozygous missense mutation in BOLA3. The causal role of the mutation was validated by lentiviral-mediated expression of the mitochondrial isoform of wildtype BOLA3 in patient fibroblasts, which lead to an increase of both residual enzyme activities and lipoic acid levels. Our results suggest that BOLA3 plays a crucial role in the biogenesis of iron-sulfur clusters necessary for proper function of respiratory chain and 2-oxoacid dehydrogenase complexes. We conclude that broad sequencing approaches combined with appropriate prioritization filters and experimental validation enable efficient molecular diagnosis and have the potential to discover new disease loci.
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Enfermedades Mitocondriales/genética , Mutación Missense , Proteínas/genética , Secuencia de Aminoácidos , Transporte de Electrón/genética , Femenino , Fibroblastos/metabolismo , Homocigoto , Humanos , Recién Nacido , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales , Datos de Secuencia Molecular , Fosforilación Oxidativa , Complejo Piruvato Deshidrogenasa/genética , Hermanos , Ácido Tióctico/metabolismoRESUMEN
BACKGROUND: Mitochondrial complex I deficiency is the most common cause of mitochondrial disease in childhood. Identification of the molecular basis is difficult given the clinical and genetic heterogeneity. Most patients lack a molecular definition in routine diagnostics. METHODS: A large-scale mutation screen of 75 candidate genes in 152 patients with complex I deficiency was performed by high-resolution melting curve analysis and Sanger sequencing. The causal role of a new disease allele was confirmed by functional complementation assays. The clinical phenotype of patients carrying mutations was documented using a standardised questionnaire. RESULTS: Causative mutations were detected in 16 genes, 15 of which had previously been associated with complex I deficiency: three mitochondrial DNA genes encoding complex I subunits, two mitochondrial tRNA genes and nuclear DNA genes encoding six complex I subunits and four assembly factors. For the first time, a causal mutation is described in NDUFB9, coding for a complex I subunit, resulting in reduction in NDUFB9 protein and both amount and activity of complex I. These features were rescued by expression of wild-type NDUFB9 in patient-derived fibroblasts. CONCLUSION: Mutant NDUFB9 is a new cause of complex I deficiency. A molecular diagnosis related to complex I deficiency was established in 18% of patients. However, most patients are likely to carry mutations in genes so far not associated with complex I function. The authors conclude that the high degree of genetic heterogeneity in complex I disorders warrants the implementation of unbiased genome-wide strategies for the complete molecular dissection of mitochondrial complex I deficiency.
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Genes Mitocondriales , Enfermedades Mitocondriales/genética , Mutación , NADH Deshidrogenasa/genética , Análisis Mutacional de ADN , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/genética , Heterogeneidad Genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Enfermedades Mitocondriales/diagnóstico , NADH Deshidrogenasa/metabolismo , FenotipoRESUMEN
Under certain conditions or at certain stages of the disease course, multiple sclerosis (MS) and mitochondrial disorder (MID) may be differential diagnoses and thus may be confused with each other. In a 30 years old female MS was diagnosed at age 16 year upon recurrent sensory disturbances of the right lower leg, an "inflammatory" cerebrospinal fluid, and a cerebral MRI with multiple non-enhancing white matter lesions. Steroids were repeatedly given but because of rapid deterioration treatment was switched to interferon and mitoxantrone, without improvement. Fourteen years after onset the patient additionally presented with a history of rhabdomyolysis, hypothyroidism, ophthalmoparesis, anarthria, tetraspasticity, tetraparesis, and joint contractures. After MID had been diagnosed in her mother she was re-evaluated and elevated resting lactate, axonal polyneuropathy, and empty sella were additionally found. Muscle biopsy revealed myophagy, fat deposition, and type-II predominance, and biochemical investigations showed a deficiency of complex I and IV of the respiratory chain. MID was diagnosed also in the index patient. It is concluded that even if CSF investigations or imaging studies suggest MS, differentials such as MIDs need to be excluded before prescribing medication possibly toxic to a MID. An "inflammatory CSF" may also occur in MIDs.
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Enfermedades Mitocondriales/diagnóstico , Esclerosis Múltiple/diagnóstico , Adulto , Edad de Inicio , Encéfalo/patología , Diagnóstico Diferencial , Femenino , Humanos , Inmunoglobulina G/líquido cefalorraquídeo , Interferón beta/uso terapéutico , Leucoencefalopatías/patología , Imagen por Resonancia Magnética , Enfermedades Mitocondriales/líquido cefalorraquídeo , Enfermedades Mitocondriales/patología , Esclerosis Múltiple/líquido cefalorraquídeo , Esclerosis Múltiple/patología , Músculo Esquelético/patología , Examen Neurológico , Parálisis/etiología , Parestesia/etiología , Trastornos de la Sensación/etiologíaRESUMEN
OBJECTIVES: SARS-CoV-2 rapid antigen tests (RAT) provide fast identification of infectious patients when RT-PCR results are not immediately available. We aimed to develop a prediction model for identification of false negative (FN) RAT results. METHODS: In this multicenter trial, patients with documented paired results of RAT and RT-PCR between October 1st 2020 and January 31st 2021 were retrospectively analyzed regarding clinical findings. Variables included demographics, laboratory values and specific symptoms. Three different models were evaluated using Bayesian logistic regression. RESULTS: The initial dataset contained 4,076 patients. Overall sensitivity and specificity of RAT was 62.3% and 97.6%. 2,997 cases with negative RAT results (FN: 120; true negative: 2,877; reference: RT-PCR) underwent further evaluation after removal of cases with missing data. The best-performing model for predicting FN RAT results containing 10 variables yielded an area under the curve of 0.971. Sensitivity, specificity, PPV and NPV for 0.09 as cut-off value (probability for FN RAT) were 0.85, 0.99, 0.7 and 0.99. CONCLUSION: FN RAT results can be accurately identified through ten routinely available variables. Implementation of a prediction model in addition to RAT testing in clinical care can provide decision guidance for initiating appropriate hygiene measures and therefore helps avoiding nosocomial infections.
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COVID-19 , SARS-CoV-2 , Teorema de Bayes , Sector de Atención de Salud , Humanos , Modelos Estadísticos , Pronóstico , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
A rapid, simple and robust HPLC-MS/MS method for simultaneous determination of immunosuppressants Cyclosporine A, Tacrolimus, Sirolimus and Everolimus has been developed and validated. Sample of whole blood with volume of 50⯵L was prepared by a protein precipitation with methanol and 0.5â¯mol. L-1 ZnSO4. Chromatographic separation was achieved on a Phenyl-Hexyl column by a gradient elution using 20â¯mmol.L-1 ammonium formate/0.1% (v/v) formic acid in water (mobile phase A) and 20â¯mmol.L-1 ammonium formate/0.1% (v/v) formic acid in methanol (mobile phase B) with flow rate 1â¯mL.min-1. The run time was 3.5â¯min. Electrospray ionization and multiple reaction monitoring was used. The lower limit of quantification (LLOQ) was set at 0.5⯵g.L-1 for Tacrolimus, Sirolimus and Everolimus and 5⯵g.L-1 for Cyclosporine A. The method demonstrated adequate accuracy and precision with sufficient linearity range.
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Cromatografía Líquida de Alta Presión/métodos , Ciclosporina/sangre , Monitoreo de Drogas/métodos , Macrólidos/sangre , Espectrometría de Masas en Tándem/métodos , Ciclosporina/química , Ciclosporina/aislamiento & purificación , Humanos , Límite de Detección , Modelos Lineales , Macrólidos/química , Macrólidos/aislamiento & purificación , Reproducibilidad de los ResultadosRESUMEN
Mild citrullinemia is an allelic variant of classical citrullinemia type I also caused by deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS). Affected patients comprise a biochemical but no clinical phenotype. However, there is no reliable parameter allowing conclusions regarding the course of the disorder or its type of manifestation. The aim of this study was to test the importance of varying levels of ASS residual activities for the severity at diagnosis. Bacterial in vitro expression studies allowed the enzymatic analysis of purified wild-type and the mutant ASS proteins p.Ala118Thr (c.352G>A), p.Trp179Arg (c.535T>C), p.Val263Met (c.787G>A), p.Arg265Cys (c.793C>T), p.Met302Val (c.904A>G), p.Gly324Ser (c.970G>A), p.Gly362Val (c.1085G>T), and p.Gly390Arg (c.1168G>A). In the chosen system, classical mutations do not show any significant enzymatic activity, whereas mutations associated with a mild course yield significant ASS activity levels. The mutation p.Ala118Thr (c.352G>A) impresses by a high residual activity (62%) but a severe reduction of affinity toward the substrates citrulline and aspartate. This mutation was identified in a hitherto healthy female adult with no history of known citrullinemia who had died during the postpartum period from hyperammonemic coma. The results of this study suggest that even a high level of residual ASS activity is not a reliable prognostic marker for an uneventful clinical course. Determination of ASS residual activities, therefore, cannot help in anticipating the risk of metabolic derangement. This study should guide clinicians as well as patients with mild citrullinemia toward a lifelong awareness of the disorder.
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Argininosuccinato Sintasa/genética , Citrulinemia/genética , Adulto , Argininosuccinato Sintasa/metabolismo , Citrulinemia/diagnóstico , ADN Complementario/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Expresión Génica , Variación Genética , Genotipo , Humanos , MutaciónAsunto(s)
NADH Deshidrogenasa/genética , Degeneración Estriatonigral/congénito , Niño , Preescolar , Complejo I de Transporte de Electrón , Femenino , Predisposición Genética a la Enfermedad , Homocigoto , Humanos , Masculino , Mutación Missense , Linaje , Hermanos , Degeneración Estriatonigral/genéticaRESUMEN
BACKGROUND: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. RESULTS: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. CONCLUSIONS: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.
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Acidosis/genética , Acidosis/metabolismo , Acil-CoA Deshidrogenasa/deficiencia , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Debilidad Muscular/genética , Debilidad Muscular/metabolismo , Riboflavina/uso terapéutico , Acidosis/patología , Actividades Cotidianas , Acil-CoA Deshidrogenasa/genética , Acil-CoA Deshidrogenasa/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/patología , Cardiomiopatía Hipertrófica/patología , Complejo I de Transporte de Electrón/metabolismo , Femenino , Humanos , Masculino , Enfermedades Mitocondriales/patología , Debilidad Muscular/tratamiento farmacológico , Debilidad Muscular/patología , PronósticoRESUMEN
OBJECTIVE: To evaluate the clinical use of Homocysteine-Primavette, a new blood collection medium for total homocysteine (tHcy) assay. METHODS: The agreement between baseline tHcy and tHcy in stabilized samples (40 h) was assessed for FPIA, HPLC, GC-MS, LC-MS, and ICL. RESULTS: tHcy concentrations in whole blood were stable for 40 h in Hcy-Primavette tubes. CONCLUSION: Primavette tubes are a good alternative for the accurate tHcy measurement and no readjustment of reference intervals is needed.
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Recolección de Muestras de Sangre/instrumentación , Homocisteína/sangre , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Ácido Edético/farmacología , Inmunoensayo de Polarización Fluorescente , Cromatografía de Gases y Espectrometría de Masas , Humanos , Mediciones Luminiscentes , Espectrometría de MasasRESUMEN
OBJECTIVES: Combined complex I+IV deficiency has rarely been reported to manifest with the involvement of the respiratory muscles. CASE REPORT: A 45y male was admitted for hypercapnia due to muscular respiratory insufficiency. He required intubation and mechanical ventilation. He had a previous history of ophthalmoparesis since age 6y, ptosis since age 23y, and anterocollis since at least age 40y. Muscle biopsy from the right deltoid muscle at age 41y was indicative of mitochondrial myopathy. Biochemical investigations revealed a combined complex I+IV defect. Respiratory insufficiency was attributed to mitochondrial myopathy affecting not only the extra-ocular and the axial muscles but also the shoulder girdle and respiratory muscles. In addition to myopathy, he had mitochondrial neuropathy, abnormal EEG, and elevated CSF-protein. Possibly, this is why a single cycle of immunoglobulins was somehow beneficial. For muscular respiratory insufficiency he required tracheostomy and was scheduled for long-term intermittent positive pressure ventilation. CONCLUSION: Mitochondrial myopathy due to a combined complex I+IV defect with predominant affection of the extra-ocular muscles may progress to involvement of the limb-girdle, axial and respiratory muscles resulting in muscular respiratory insufficiency. In patients with mitochondrial myopathy, neuropathy and elevated cerebrospinal fluid protein, immunoglobulins may be beneficial even for respiratory functions.
RESUMEN
Disorders of the mitochondrial energy metabolism are clinically and genetically heterogeneous. An increasingly recognized subgroup is caused by defective mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, with defects in 13 genes being linked to human disease to date. Mutations in three of them, NFU1, BOLA3, and IBA57, affect the assembly of mitochondrial [4Fe-4S] proteins leading to an impairment of diverse mitochondrial metabolic pathways and ATP production. Patients with defects in these three genes present with lactic acidosis, hyperglycinemia, and reduced activities of respiratory chain complexes I and II, the four lipoic acid-dependent 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). To date, five different NFU1 pathogenic variants have been reported in 15 patients from 12 families. We report on seven new patients from five families carrying compound heterozygous or homozygous pathogenic NFU1 mutations identified by candidate gene screening and exome sequencing. Six out of eight different disease alleles were novel and functional studies were performed to support the pathogenicity of five of them. Characteristic clinical features included fatal infantile encephalopathy and pulmonary hypertension leading to death within the first 6 months of life in six out of seven patients. Laboratory investigations revealed combined defects of pyruvate dehydrogenase complex (five out of five) and respiratory chain complexes I and II+III (four out of five) in skeletal muscle and/or cultured skin fibroblasts as well as increased lactate (five out of six) and glycine concentration (seven out of seven). Our study contributes to a better definition of the phenotypic spectrum associated with NFU1 mutations and to the diagnostic workup of future patients.
RESUMEN
OBJECTIVES: Loss of consciousness may be due to neurological or cardiac involvement in mitochondrial disease, and is often difficult to attribute to either cause, as in the following case. CASE REPORT: A 67-year-old man with hypertension, diabetes, elevated serum creatine kinase, glaucoma, optic atrophy, and vertigo had experienced recurrent losses of consciousness since 63 years of age. Diagnostic work-up revealed paroxysmal supraventricular arrhythmias, hyperlipidemia, steatosis hepatis, renal insufficiency, polyneuropathy, first-degree atrio-ventricular block, orthostasis, and cataract. From the age of 66 years, he developed tonic-clonic seizures. Electrocardiography loop recording showed some losses of consciousness as associated with supraventricular tachycardias and others with epileptic activity or arterial hypotension. Neurological investigations and muscle biopsy were indicative of mitochondrial disease with multisystem involvement. Losses of consciousness disappeared after catheter ablation and treatment with levetiracetam. CONCLUSION: Recurrent loss of consciousness in mitochondrial disease may not only be due to arrhythmias but also seizure activity, or autonomic neuropathy. Arrhythmias, seizures, and polyneuropathy may have a common underlying cause affecting various tissues.
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Epilepsia Tónico-Clónica/etiología , Enfermedades Mitocondriales/complicaciones , Síncope/etiología , Taquicardia Supraventricular/etiología , Anciano , Anticonvulsivantes/uso terapéutico , Enfermedades del Sistema Nervioso Autónomo/etiología , Biopsia , Ablación por Catéter , Electrocardiografía , Epilepsia Tónico-Clónica/diagnóstico , Epilepsia Tónico-Clónica/tratamiento farmacológico , Humanos , Levetiracetam , Masculino , Enfermedades Mitocondriales/diagnóstico , Piracetam/análogos & derivados , Piracetam/uso terapéutico , Recurrencia , Síncope/prevención & control , Taquicardia Supraventricular/diagnóstico , Taquicardia Supraventricular/cirugía , Resultado del TratamientoRESUMEN
An isolated defect of respiratory chain complex I activity is a frequent biochemical abnormality in mitochondrial disorders. Despite intensive investigation in recent years, in most instances, the molecular basis underpinning complex I defects remains unknown. We report whole-exome sequencing of a single individual with severe, isolated complex I deficiency. This analysis, followed by filtering with a prioritization of mitochondrial proteins, led us to identify compound heterozygous mutations in ACAD9, which encodes a poorly understood member of the mitochondrial acyl-CoA dehydrogenase protein family. We demonstrated the pathogenic role of the ACAD9 variants by the correction of the complex I defect on expression of the wildtype ACAD9 protein in fibroblasts derived from affected individuals. ACAD9 screening of 120 additional complex I-defective index cases led us to identify two additional unrelated cases and a total of five pathogenic ACAD9 alleles.
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Acil-CoA Deshidrogenasas/genética , Complejo I de Transporte de Electrón/deficiencia , Exones/genética , Mutación/genética , Análisis de Secuencia de ADN , Acil-CoA Deshidrogenasas/química , Secuencia de Aminoácidos , Línea Celular , Niño , Preescolar , Complejo I de Transporte de Electrón/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Prueba de Complementación Genética , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Riboflavina/farmacología , Transducción GenéticaRESUMEN
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1-NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are "ciliopathies". Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
Asunto(s)
Aminopeptidasas/metabolismo , Enfermedades Genéticas Congénitas/enzimología , Riñón/enzimología , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Insuficiencia Renal/enzimología , Aminopeptidasas/genética , Animales , Centrosoma/enzimología , Centrosoma/patología , Mapeo Cromosómico/métodos , Cilios/enzimología , Cilios/genética , Cilios/patología , Familia , Femenino , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/patología , Estudio de Asociación del Genoma Completo/métodos , Humanos , Riñón/patología , Masculino , Mitocondrias/patología , Proteínas Mitocondriales/genética , Ratas , Ratas Sprague-Dawley , Insuficiencia Renal/genética , Insuficiencia Renal/patología , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
OBJECTIVE: High blood concentrations of bilirubin are toxic to the brain and may cause kernicterus. Therefore, determination of bilirubin levels is performed for many newborns, and several different methods are available. We compared 9 frequently used methods for bilirubin determination among newborns under routine conditions, to define their sequence of use. METHODS: In a prospective study, bilirubin concentrations were determined with 9 different methods, ie, 3 skin test devices, 3 nonchemical photometric devices (including 2 blood gas analyzers), and 3 laboratory analyzers. RESULTS: A total of 124 samples were obtained. All 3 laboratory methods showed very strong correlations with each other, and their means were used as comparison values. To these comparison values, the skin test devices had correlation coefficients between 0.961 and 0.966, and the nonchemical photometric devices between 0.980 and 0.994. Bland-Altman plots demonstrated good agreement with the comparison values for all nonchemical photometric devices. All skin test devices and 1 nonchemical photometric device underestimated bilirubin levels, particularly at high concentrations. CONCLUSIONS: In the routine care of newborns, the first method for bilirubin testing should be a skin test. If the skin test result exceeds 200 micromol/L and other analytes are to be determined with a nonchemical photometric device, then bilirubin can be included in this analysis and the result trusted up to 250 micromol/L. If the skin test result exceeds 200 micromol/L and only bilirubin concentrations are needed, then a standard laboratory method is the first choice, to avoid repeated blood sampling. Bilirubin concentrations from nonchemical photometric devices that exceed 250 micromol/L should be confirmed with standard laboratory methods.