RESUMEN
A 6-yr-old female orangutan presented with a history of dark urine that turned brown upon standing since birth. Repeated routine urinalysis and urine culture were unremarkable. Urine organic acid analysis showed elevation in homogentisic acid consistent with alkaptonuria. Sequence analysis identified a homozygous missense variant, c.1081G>A (p.Gly361Arg), of the homogentisate 1,2-dioxygenase (HGD) gene. Familial studies, molecular modeling, and comparison to human variant databases support this variant as the underlying cause of alkaptonuria in this orangutan. This is the first report of molecular confirmation of alkaptonuria in a nonhuman primate.
Asunto(s)
Alcaptonuria , Pongo abelii , Animales , Humanos , Femenino , Alcaptonuria/diagnóstico , Alcaptonuria/genética , Pongo abelii/genética , Ácido Homogentísico , Mutación Missense , HomocigotoRESUMEN
The peroxisome is an essential eukaryotic organelle with diverse metabolic functions. Inherited peroxisomal disorders are associated with a wide spectrum of clinical outcomes and are broadly divided into two classes, those impacting peroxisome biogenesis (PBD) and those impacting specific peroxisomal factors. Prior studies have indicated a role for acylcarnitine testing in the diagnosis of some peroxisomal diseases through the detection of long chain dicarboxylic acylcarnitine abnormalities (C16-DC and C18-DC). However, there remains limited independent corroboration of these initial findings and acylcarnitine testing for peroxisomal diseases has not been widely adopted in clinical laboratories. To explore the utility of acylcarnitine testing in the diagnosis of peroxisomal disorders we applied a LC-MS/MS acylcarnitine method to study a heterogenous clinical sample set (n = 598) that included residual plasma specimens from nineteen patients with PBD caused by PEX1 or PEX6 deficiency, ranging in severity from lethal neonatal onset to mild late onset forms. Multiple dicarboxylic acylcarnitines were significantly elevated in PBD patients including medium to long chain (C8-DC to C18-DC) species as well as previously undescribed elevations of malonylcarnitine (C3-DC) and very long chain dicarboxylic acylcarnitines (C20-DC and C22-DC). The best performing plasma acylcarnitine biomarkers, C20-DC and C22-DC, were detected at elevated levels in 100% and 68% of PBD patients but were rarely elevated in patients that did not have a PBD. We extended our analysis to residual newborn screening blood spot cards and were able to detect dicarboxylic acylcarnitine abnormalities in a newborn with a PBD caused by PEX6 deficiency. Similar to prior studies, we failed to detect substantial dicarboxylic acylcarnitine abnormalities in blood spot cards from patients with x-linked adrenoleukodystrophy (x-ald) indicating that these biomarkers may have utility in quickly narrowing the differential diagnosis in patients with a positive newborn screen for x-ald. Overall, our study identifies widespread dicarboxylic acylcarnitine abnormalities in patients with PBD and highlights key acylcarnitine biomarkers for the detection of this class of inherited metabolic disease.
Asunto(s)
Adrenoleucodistrofia , Trastorno Peroxisomal , Recién Nacido , Humanos , Adrenoleucodistrofia/diagnóstico , Adrenoleucodistrofia/genética , Cromatografía Liquida , Espectrometría de Masas en Tándem , Trastorno Peroxisomal/diagnóstico , Trastorno Peroxisomal/genética , Biomarcadores , ATPasas Asociadas con Actividades Celulares Diversas , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (VLCADD) is a relatively common inborn error of metabolism, but due to difficulty in accurately predicting affected status through newborn screening, molecular confirmation of the causative variants by sequencing of the ACADVL gene is necessary. Although the ACMG/AMP guidelines have helped standardize variant classification, ACADVL variant classification remains disparate due to a phenotype that can be nonspecific, the possibility of variants that produce late-onset disease, and relatively high carrier frequency, amongst other challenges. Therefore, an ACADVL-specific variant curation expert panel (VCEP) was created to facilitate the specification of the ACMG/AMP guidelines for VLCADD. We expect these guidelines to help streamline, increase concordance, and expedite the classification of ACADVL variants.
Asunto(s)
Errores Innatos del Metabolismo Lipídico , Enfermedades Mitocondriales , Enfermedades Musculares , Humanos , Recién Nacido , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Síndromes Congénitos de Insuficiencia de la Médula Ósea/genética , Pruebas Genéticas , Variación Genética , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Enfermedades Mitocondriales/genética , Enfermedades Musculares/genéticaRESUMEN
WWOX biallelic loss-of-function pathogenic single nucleotide variants (SNVs) and copy number variants (CNVs) including exonic deletions and duplications cause WWOX-related epileptic encephalopathy (WOREE) syndrome. This disorder is characterized by refractory epilepsy, axial hypotonia, peripheral hypertonia, progressive microcephaly, and premature death. Here we report five patients with WWOX biallelic predicted null variants identified by exome sequencing (ES), genome sequencing (GS), and/or chromosomal microarray analysis (CMA). SNVs and intragenic deletions of one or more exons were commonly reported in WOREE syndrome patients which made the genetic diagnosis challenging and required a combination of different diagnostic technologies. These patients presented with severe, developmental and epileptic encephalopathy (DEE), and other cardinal features consistent with WOREE syndrome. This report expands the clinical phenotype associated with this condition, including failure to thrive in most patients and epilepsy that responded to a ketogenic diet in three patients. Dysmorphic features and abnormal prenatal findings were not commonly observed. Additionally, recurrent pancreatitis and sensorineural hearing loss each were observed in single patients. In summary, these phenotypic features broaden the clinical spectrum of WOREE syndrome.
Asunto(s)
Encefalopatías , Epilepsia Generalizada , Epilepsia , Síndromes Epilépticos , Femenino , Embarazo , Humanos , Epilepsia/diagnóstico , Epilepsia/genética , Síndromes Epilépticos/genética , Encefalopatías/genética , Epilepsia Generalizada/genética , Exones , Oxidorreductasa que Contiene Dominios WW/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
[This corrects the article DOI: 10.1371/journal.pcbi.1008550.].
RESUMEN
We consider the following general family of algorithmic problems that arises in transcriptomics, metabolomics and other fields: given a weighted graph G and a subset of its nodes S, find subsets of S that show significant connectedness within G. A specific solution to this problem may be defined by devising a scoring function, the Maximum Clique problem being a classic example, where S includes all nodes in G and where the score is defined by the size of the largest subset of S fully connected within G. Major practical obstacles for the plethora of algorithms addressing this type of problem include computational efficiency and, particularly for more complex scores which take edge weights into account, the computational cost of permutation testing, a statistical procedure required to obtain a bound on the p-value for a connectedness score. To address these problems, we developed CTD, "Connect the Dots", a fast algorithm based on data compression that detects highly connected subsets within S. CTD provides information-theoretic upper bounds on p-values when S contains a small fraction of nodes in G without requiring computationally costly permutation testing. We apply the CTD algorithm to interpret multi-metabolite perturbations due to inborn errors of metabolism and multi-transcript perturbations associated with breast cancer in the context of disease-specific Gaussian Markov Random Field networks learned directly from respective molecular profiling data.
Asunto(s)
Algoritmos , Perfilación de la Expresión Génica/métodos , Teoría de la Información , Metabolómica/métodos , Gráficos por Computador , Humanos , Metaboloma/genética , Transcriptoma/genéticaRESUMEN
Acylcarnitine analysis is a useful test for identifying patients with inborn errors of mitochondrial fatty acid ß-oxidation and certain organic acidemias. Plasma is routinely used in the diagnostic workup of symptomatic patients. Urine analysis of targeted acylcarnitine species may be helpful in the diagnosis of glutaric acidemia type I and other disorders in which polar acylcarnitine species accumulate. For newborn screening applications, dried blood spot acylcarnitine analysis can be performed as a multiplex assay with other analytes, including amino acids, succinylacetone, guanidinoacetate, creatine, and lysophosphatidylcholines. Tandem mass spectrometric methodology, established more than 30 years ago, remains a valid approach for acylcarnitine analysis. The method involves flow-injection analysis of esterified or underivatized acylcarnitines species and detection using a precursor-ion scan. Alternative methods utilize liquid chromatographic separation of isomeric and isobaric species and/or detection by selected reaction monitoring. These technical standards were developed as a resource for diagnostic laboratory practices in acylcarnitine analysis, interpretation, and reporting.
Asunto(s)
Genética Médica , Laboratorios , Carnitina/análogos & derivados , Genómica , Humanos , Recién Nacido , Estados UnidosRESUMEN
The posttranslational addition of small ubiquitin-like modifier (SUMO) is an essential protein modification in plants that provides protection against numerous environmental challenges. Ligation is accomplished by a small set of SUMO ligases, with the SAP-MIZ domain-containing SIZ1 and METHYL METHANESULFONATE-SENSITIVE21 (MMS21) ligases having critical roles in stress protection and DNA endoreduplication/repair, respectively. To help identify their corresponding targets in Arabidopsis thaliana, we used siz1 and mms21 mutants for proteomic analyses of SUMOylated proteins enriched via an engineered SUMO1 isoform suitable for mass spectrometric studies. Through multiple data sets from seedlings grown at normal temperatures or exposed to heat stress, we identified over 1000 SUMO targets, most of which are nuclear localized. Whereas no targets could be assigned to MMS21, suggesting that it modifies only a few low abundance proteins, numerous targets could be assigned to SIZ1, including major transcription factors, coactivators/repressors, and chromatin modifiers connected to abiotic and biotic stress defense, some of which associate into multisubunit regulatory complexes. SIZ1 itself is also a target, but studies with mutants protected from SUMOylation failed to uncover a regulatory role. The catalog of SIZ1 substrates indicates that SUMOylation by this ligase provides stress protection by modifying a large array of key nuclear regulators.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Respuesta al Choque Térmico/genética , Respuesta al Choque Térmico/fisiología , Proteómica/métodos , Plantones/genética , Plantones/metabolismo , Sumoilación/genética , Sumoilación/fisiología , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
PURPOSE: Untargeted metabolomic analysis is increasingly being used in the screening and management of individuals with inborn errors of metabolism (IEM). We aimed to test whether untargeted metabolomic analysis in plasma might be useful for monitoring the disease course and management of urea cycle disorders (UCDs). METHODS: Untargeted mass spectrometry-based metabolomic analysis was used to generate z-scores for more than 900 metabolites in plasma from 48 individuals with various UCDs. Pathway analysis was used to identify common pathways that were perturbed in each UCD. RESULTS: Our metabolomic analysis in plasma identified multiple potentially neurotoxic metabolites of arginine in arginase deficiency and, thus, may have utility in monitoring the efficacy of treatment in arginase deficiency. In addition, we were also able to detect multiple biochemical perturbations in all UCDs that likely reflect clinical management, including metabolite alterations secondary to dietary and medication management. CONCLUSION: In addition to utility in screening for IEM, our results suggest that untargeted metabolomic analysis in plasma may be beneficial for monitoring efficacy of clinical management and off-target effects of medications in UCDs and potentially other IEM.
Asunto(s)
Biomarcadores/sangre , Errores Innatos del Metabolismo/sangre , Metabolómica , Trastornos Innatos del Ciclo de la Urea/sangre , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Espectrometría de Masas , Redes y Vías Metabólicas/genética , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/patología , Urea/metabolismo , Trastornos Innatos del Ciclo de la Urea/genética , Trastornos Innatos del Ciclo de la Urea/patología , Adulto JovenRESUMEN
PURPOSE: Peroxisome biogenesis disorders-Zellweger spectrum disorders (PBD-ZSD) are metabolic diseases with multisystem manifestations. Individuals with PBD-ZSD exhibit impaired peroxisomal biochemical functions and have abnormal levels of peroxisomal metabolites, but the broader metabolic impact of peroxisomal dysfunction and the utility of metabolomic methods is unknown. METHODS: We studied 19 individuals with clinically and molecularly characterized PBD-ZSD. We performed both quantitative peroxisomal biochemical diagnostic studies in parallel with untargeted small molecule metabolomic profiling in plasma samples with detection of >650 named compounds. RESULTS: The cohort represented intermediate to mild PBD-ZSD subjects with peroxisomal biochemical alterations on targeted analysis. Untargeted metabolomic profiling of these samples revealed elevations in pipecolic acid and long-chain lysophosphatidylcholines, as well as an unanticipated reduction in multiple sphingomyelin species. These sphingomyelin reductions observed were consistent across the PBD-ZSD samples and were rare in a population of >1,000 clinical samples. Interestingly, the pattern or "PBD-ZSD metabolome" was more pronounced in younger subjects suggesting studies earlier in life reveal larger biochemical changes. CONCLUSION: Untargeted metabolomics is effective in detecting mild to intermediate cases of PBD-ZSD. Surprisingly, dramatic reductions in plasma sphingomyelin are a consistent feature of the PBD-ZSD metabolome. The use of metabolomics in PBD-ZSD can provide insight into novel biomarkers of disease.
Asunto(s)
Biomarcadores/sangre , Enfermedades por Almacenamiento Lisosomal/sangre , Trastorno Peroxisomal/sangre , Síndrome de Zellweger/sangre , Adolescente , Adulto , Preescolar , Estudios de Cohortes , Femenino , Humanos , Enfermedades por Almacenamiento Lisosomal/genética , Enfermedades por Almacenamiento Lisosomal/patología , Masculino , Proteínas de la Membrana , Metabolómica/métodos , Trastorno Peroxisomal/patología , Esfingomielinas/sangre , Adulto Joven , Síndrome de Zellweger/genética , Síndrome de Zellweger/patologíaRESUMEN
We sought to determine the molecular composition of human cerebrospinal fluid (CSF) and identify the biochemical pathways represented in CSF to understand the potential for untargeted screening of inborn errors of metabolism (IEMs). Biochemical profiles for each sample were obtained using an integrated metabolomics workflow comprised of four chromatographic techniques followed by mass spectrometry. Secondarily, we wanted to compare the biochemical profile of CSF with those of plasma and urine within the integrated mass spectrometric-based metabolomic workflow. Three sample types, CSF (N=30), urine (N=40) and EDTA plasma (N=31), were analyzed from retrospectively collected pediatric cohorts of equivalent age and gender characteristics. We identified 435 biochemicals in CSF representing numerous biological and chemical/structural families. Sixty-three percent (273 of 435) of the biochemicals detected in CSF also were detected in urine and plasma, another 32% (140 of 435) were detected in either plasma or urine, and 5% (22 of 435) were detected only in CSF. Analyses of several metabolites showed agreement between clinically useful assays and the metabolomics approach. An additional set of CSF and plasma samples collected from the same patient revealed correlation between several biochemicals detected in paired samples. Finally, analysis of CSF from a pediatric case with dihydropteridine reductase (DHPR) deficiency demonstrated the utility of untargeted global metabolic phenotyping as a broad assessment to screen samples from patients with undifferentiated phenotypes. The results indicate a single CSF sample processed with an integrated metabolomics workflow can be used to identify a large breadth of biochemicals that could be useful for identifying disrupted metabolic patterns associated with IEMs.
Asunto(s)
Proteínas del Líquido Cefalorraquídeo/genética , Proteínas del Líquido Cefalorraquídeo/metabolismo , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/metabolismo , Metaboloma , Metabolómica/métodos , Adolescente , Biomarcadores/sangre , Biomarcadores/orina , Proteínas del Líquido Cefalorraquídeo/análisis , Proteínas del Líquido Cefalorraquídeo/química , Niño , Preescolar , Dihidropteridina Reductasa/sangre , Dihidropteridina Reductasa/genética , Dihidropteridina Reductasa/metabolismo , Dihidropteridina Reductasa/orina , Femenino , Humanos , Lactante , Masculino , Espectrometría de Masas/métodos , Errores Innatos del Metabolismo/diagnóstico , Fenotipo , Estudios Retrospectivos , Adulto JovenAsunto(s)
Acidosis Láctica , Acidosis , Hiperamonemia , Cetosis , Acidosis Láctica/diagnóstico , Niño , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/etiologíaRESUMEN
OBJECTIVES: To test whether follow-up testing for very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency uncovers a diagnosis in patients with elevations of C14:1 and C14:2 plasma acylcarnitines after a controlled fasting study performed for clinically suspected hypoglycemia and to compare the acylcarnitine profiles from fasted patients without VLCAD deficiency vs patients with known VLCAD deficiency to determine whether metabolite testing distinguishes these groups. STUDY DESIGN: We performed a retrospective chart review and identified 17 patients with elevated C14:1 and C14:2 plasma acylcarnitine levels after a controlled fast and with testing for VLCAD deficiency (ACADVL sequencing or fibroblast fatty acid oxidation studies). The follow-up testing in all patients was inconsistent with a diagnosis of VLCAD deficiency. We compared the plasma acylcarnitine profiles from these fasted patients vs patients with VLCAD deficiency. RESULTS: C14:1/C12:1 was significantly lower (P < .001) in fasted patients vs patients with VLCAD deficiency. Metabolomics analysis performed in 2 fasted patients and 1 patient with VLCAD deficiency demonstrated evidence for up-regulated lipolysis and ß-oxidation in the fasted state. CONCLUSIONS: Elevations of plasma C14:1 and C14:2 acylcarnitines appear to be a physiologic result of lipolysis that occurs with fasting. Both metabolomics analysis and/or C14:1/C12:1 may distinguish C14:1 elevations from physiologic fasting-induced lipolysis vs VLCAD deficiency.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Carnitina/análogos & derivados , Ayuno/sangre , Errores Innatos del Metabolismo Lipídico/sangre , Errores Innatos del Metabolismo Lipídico/diagnóstico , Enfermedades Mitocondriales/sangre , Enfermedades Mitocondriales/diagnóstico , Enfermedades Musculares/sangre , Enfermedades Musculares/diagnóstico , Acil-CoA Deshidrogenasa de Cadena Larga/sangre , Adolescente , Carnitina/sangre , Niño , Preescolar , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Femenino , Humanos , Lactante , Masculino , Estudios RetrospectivosRESUMEN
Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Acil-CoA Deshidrogenasa de Cadena Larga/genética , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/genética , Tamizaje Neonatal , Alelos , Carnitina/análogos & derivados , Simulación por Computador , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Exones , Femenino , Tamización de Portadores Genéticos , Genotipo , Humanos , Hipoglucemia/etiología , Recién Nacido , Masculino , Mutación Missense , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia de ADN , Espectrometría de Masas en Tándem , Estados UnidosRESUMEN
Global metabolic profiling currently achievable by untargeted mass spectrometry-based metabolomic platforms has great potential to advance our understanding of human disease states, including potential utility in the detection of novel and known inborn errors of metabolism (IEMs). There are few studies of the technical reproducibility, data analysis methods, and overall diagnostic capabilities when this technology is applied to clinical specimens for the diagnosis of IEMs. We explored the clinical utility of a metabolomic workflow capable of routinely generating semi-quantitative z-score values for ~900 unique compounds, including ~500 named human analytes, in a single analysis of human plasma. We tested the technical reproducibility of this platform and applied it to the retrospective diagnosis of 190 individual plasma samples, 120 of which were collected from patients with a confirmed IEM. Our results demonstrate high intra-assay precision and linear detection for the majority compounds tested. Individual metabolomic profiles provided excellent sensitivity and specificity for the detection of a wide range of metabolic disorders and identified novel biomarkers for some diseases. With this platform, it is possible to use one test to screen for dozens of IEMs that might otherwise require ordering multiple unique biochemical tests. However, this test may yield false negative results for certain disorders that would be detected by a more well-established quantitative test and in its current state should be considered a supplementary test. Our findings describe a novel approach to metabolomic analysis of clinical specimens and demonstrate the clinical utility of this technology for prospective screening of IEMs.
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Biomarcadores/análisis , Errores Innatos del Metabolismo/diagnóstico , Metabolómica/métodos , Tamizaje Neonatal/métodos , Humanos , Recién Nacido , Reproducibilidad de los Resultados , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
The stress-induced attachment of small ubiquitin-like modifier (SUMO) to a diverse collection of nuclear proteins regulating chromatin architecture, transcription, and RNA biology has been implicated in protecting plants and animals against numerous environmental challenges. In order to better understand stress-induced SUMOylation, we combined stringent purification of SUMO conjugates with isobaric tag for relative and absolute quantification mass spectrometry and an advanced method to adjust for sample-to-sample variation so as to study quantitatively the SUMOylation dynamics of intact Arabidopsis seedlings subjected to stress. Inspection of 172 SUMO substrates during and after heat shock (37 °C) revealed that stress mostly increases the abundance of existing conjugates, as opposed to modifying new targets. Some of the most robustly up-regulated targets participate in RNA processing and turnover and RNA-directed DNA modification, thus implicating SUMO as a regulator of the transcriptome during stress. Many of these targets were also strongly SUMOylated during ethanol and oxidative stress, suggesting that their modification is crucial for general stress tolerance. Collectively, our quantitative data emphasize the importance of SUMO to RNA-related processes protecting plants from adverse environments.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas Nucleares/genética , ARN de Planta/genética , Plantones/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/análisis , Proteínas de Arabidopsis/metabolismo , Cromatografía Liquida , Etanol/farmacología , Respuesta al Choque Térmico , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Estrés Oxidativo , Proteómica , ARN de Planta/metabolismo , Plantones/química , Plantones/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/análisis , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Coloración y Etiquetado , Estrés Fisiológico , Sumoilación , Espectrometría de Masas en Tándem , Transcriptoma/efectos de los fármacosRESUMEN
Citrullinemia type I is a urea cycle disorder caused by autosomal recessive mutations in argininosuccinate synthetase 1 (ASS1). In the classical form of this disease, symptoms manifest during the neonatal period as progressive lethargy, poor feeding, and central nervous system depression secondary to hyperammonemia. In pregnancies involving two carrier parents, prenatal diagnosis is important for both reproductive decisions and advanced preparation for neonatal care. The current gold standard for prenatal diagnosis has been the citrulline incorporation assay in addition to DNA mutation analysis. Herein, we review our experience with prenatal diagnosis of citrullinemia type I over the span of 11 years in 41 at-risk pregnancies. During this time, we identified 15 affected fetuses using a combination of molecular and biochemical testing. Given the established limitations of both the citrulline incorporation assay as well DNA mutation analysis, we probed our data to assess the value of amniotic fluid amino acid levels in prenatal diagnosis. Previous publications have proposed using the amniotic fluid ratio of citrulline/(arginine+ornithine) in prenatal diagnosis; however, we noted that amniotic fluid arginine levels were normal in our cohort and hypothesized that the amniotic fluid citrulline/ornithine ratio may be superior. Indeed, our analyses revealed that the ratio of amniotic fluid citrulline/ornithine alone correctly distinguished affected from unaffected fetuses in all cases. During the establishment of a normal reference range we discovered significant elevations in amniotic fluid citrulline levels in at-risk pregnancies compared to the normal population even when the fetus was unaffected. This highlights the importance of using amniotic fluid from carrier mothers when setting up a normal reference range. Finally, we report our experience as one of the first centers to adopt Sanger sequencing for prospective prenatal diagnosis of citrullinemia. While this is clearly a useful tool in many cases, we encountered families for whom molecular analysis uncovered variants of unknown clinical significance or no mutation at all. Based upon these new findings, we recommend a combinatorial approach involving ASS1 sequencing and amniotic fluid citrulline/ornithine for the prenatal diagnosis of citrullinemia type I.
Asunto(s)
Citrulinemia/diagnóstico , Citrulinemia/genética , Diagnóstico Prenatal/métodos , Diagnóstico Prenatal/normas , Adulto , Líquido Amniótico/metabolismo , Argininosuccinato Sintasa/genética , Argininosuccinato Sintasa/metabolismo , Citrulina/metabolismo , Activación Enzimática , Femenino , Feto/metabolismo , Humanos , Masculino , Mutación , Embarazo , Estudios RetrospectivosRESUMEN
The covalent attachment of SUMO (small ubiquitin-like modifier) to other intracellular proteins affects a broad range of nuclear processes in yeast and animals, including chromatin maintenance, transcription, and transport across the nuclear envelope, as well as protects proteins from ubiquitin addition. Substantial increases in SUMOylated proteins upon various stresses have also implicated this modification in the general stress response. To help understand the role(s) of SUMOylation in plants, we developed a stringent method to isolate SUMO-protein conjugates from Arabidopsis thaliana that exploits a tagged SUMO1 variant that faithfully replaces the wild-type protein. Following purification under denaturing conditions, SUMOylated proteins were identified by tandem mass spectrometry from both nonstressed plants and those exposed to heat and oxidative stress. The list of targets is enriched for factors that direct SUMOylation and for nuclear proteins involved in chromatin remodeling/repair, transcription, RNA metabolism, and protein trafficking. Targets of particular interest include histone H2B, components in the LEUNIG/TOPLESS corepressor complexes, and proteins that control histone acetylation and DNA methylation, which affect genome-wide transcription. SUMO attachment site(s) were identified in a subset of targets, including SUMO1 itself to confirm the assembly of poly-SUMO chains. SUMO1 also becomes conjugated with ubiquitin during heat stress, thus connecting these two posttranslational modifications in plants. Taken together, we propose that SUMOylation represents a rapid and global mechanism for reversibly manipulating plant chromosomal functions, especially during environmental stress.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sitios de Unión , Núcleo Celular/metabolismo , Cromatografía de Afinidad , Variación Genética , Calor , Plantas Modificadas Genéticamente , Proteómica , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Estrés Fisiológico , Espectrometría de Masas en Tándem , UbiquitinaciónRESUMEN
Lysine succinylation is a subtype of protein acylation associated with metabolic regulation of succinyl-CoA in the tricarboxylic acid cycle. Deficiency of succinyl-CoA synthetase (SCS), the tricarboxylic acid cycle enzyme catalyzing the interconversion of succinyl-CoA to succinate, results in mitochondrial encephalomyopathy in humans. This report presents a conditional forebrain-specific knockout (KO) mouse model of Sucla2, the gene encoding the ATP-specific beta isoform of SCS, resulting in postnatal deficiency of the entire SCS complex. Results demonstrate that accumulation of succinyl-CoA in the absence of SCS leads to hypersuccinylation within the murine cerebral cortex. Specifically, increased succinylation is associated with functionally significant reduced activity of respiratory chain complex I and widescale alterations in chromatin landscape and gene expression. Integrative analysis of the transcriptomic data also reveals perturbations in regulatory networks of neuronal transcription in the KO forebrain. Together, these findings provide evidence that protein succinylation plays a significant role in the pathogenesis of SCS deficiency.
Asunto(s)
Mitocondrias , Succinato-CoA Ligasas , Humanos , Animales , Ratones , Mitocondrias/metabolismo , Acilcoenzima A/metabolismo , Succinato-CoA Ligasas/genética , Succinato-CoA Ligasas/metabolismo , Ratones NoqueadosRESUMEN
Acylcarnitines are intermediate metabolites of the mitochondria that serve as biomarkers for inherited disorders of fatty acid oxidation and amino acid metabolism. The prevailing clinical method used to quantify acylcarnitines involves flow-injection tandem mass spectrometry, an approach with a number of limitations; foremost the inability to separate and therefore distinguish key isobaric acylcarnitine species. To address these issues, we report a clinically validated liquid chromatography tandem mass spectrometry method to quantify acylcarnitines, free carnitine, and carnitine metabolic intermediates in human plasma. Importantly, this method resolves clinically relevant isobaric and isomeric acylcarnitine species in a single 22 min analysis without the use of ion pairing or derivatization reagents. This unique combination of features is not achievable by existing acylcarnitine methods and is made possible by the use of a novel mixed-mode chromatographic separation. Further clinical validation studies demonstrate excellent limits of quantification, linearity, accuracy, and inter-assay precision for analyses of 38 different calibrated analytes. An additional 28 analytes are semi-quantitatively analyzed using surrogate calibrators. The study of residual patient specimens confirms the clinical utility of this method and suggests expanded applicability to the diagnosis of peroxisomal disorders. In summary, we report a clinically validated acylcarnitine method that utilizes a novel mixed-mode chromatographic separation to provide a number of advantages in terms of specificity, accuracy, sample preparation time, and clinical utility.