RESUMEN
Accurate determination of the clinical significance of genetic variants is critical to the integration of genomics in medicine. To facilitate this process, the NIH-funded Clinical Genome Resource (ClinGen) has assembled Variant Curation Expert Panels (VCEPs), groups of experts and biocurators which provide gene- and disease- specifications to the American College of Medical Genetics & Genomics and Association for Molecular Pathology's (ACMG/AMP) variation classification guidelines. With the goal of classifying the clinical significance of GAA variants in Pompe disease (Glycogen storage disease, type II), the ClinGen Lysosomal Diseases (LD) VCEP has specified the ACMG/AMP criteria for GAA. Variant classification can play an important role in confirming the diagnosis of Pompe disease as well as in the identification of carriers. Furthermore, since the inclusion of Pompe disease on the Recommended Uniform Screening Panel (RUSP) for newborns in the USA in 2015, the addition of molecular genetic testing has become an important component in the interpretation of newborn screening results, particularly for asymptomatic individuals. To date, the LD VCEP has submitted classifications and supporting data on 243 GAA variants to public databases, specifically ClinVar and the ClinGen Evidence Repository. Here, we describe the ACMG/AMP criteria specification process for GAA, an update of the GAA-specific variant classification guidelines, and comparison of the ClinGen LD VCEP's GAA variant classifications with variant classifications submitted to ClinVar. The LD VCEP has added to the publicly available knowledge on the pathogenicity of variants in GAA by increasing the number of expert-curated GAA variants present in ClinVar, and aids in resolving conflicting classifications and variants of uncertain clinical significance.
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Variación Genética , Enfermedad del Almacenamiento de Glucógeno Tipo II , Recién Nacido , Humanos , Estados Unidos , Pruebas Genéticas/métodos , Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Enfermedad del Almacenamiento de Glucógeno Tipo II/genética , Genoma Humano , Genómica/métodosRESUMEN
[This corrects the article DOI: 10.1371/journal.pbio.1002197.].
RESUMEN
Inborn errors of metabolism (IEM) involving the non-oxidative pentose phosphate pathway (PPP) include the two relatively rare conditions, transketolase deficiency and transaldolase deficiency, both of which can be difficult to diagnosis given their non-specific clinical presentations. Current biochemical testing approaches require an index of suspicion to consider targeted urine polyol testing. To determine whether a broad-spectrum biochemical test could accurately identify a specific metabolic pattern defining IEMs of the non-oxidative PPP, we employed the use of clinical metabolomic profiling as an unbiased novel approach to diagnosis. Subjects with molecularly confirmed IEMs of the PPP were included in this study. Targeted quantitative analysis of polyols in urine and plasma samples was accomplished with chromatography and mass spectrometry. Semi-quantitative unbiased metabolomic analysis of urine and plasma samples was achieved by assessing small molecules via liquid chromatography and high-resolution mass spectrometry. Results from untargeted and targeted analyses were then compared and analyzed for diagnostic acuity. Two siblings with transketolase (TKT) deficiency and three unrelated individuals with transaldolase (TALDO) deficiency were identified for inclusion in the study. For both IEMs, targeted polyol testing and untargeted metabolomic testing on urine and/or plasma samples identified typical perturbations of the respective disorder. Additionally, untargeted metabolomic testing revealed elevations in other PPP metabolites not typically measured with targeted polyol testing, including ribonate, ribose, and erythronate for TKT deficiency and ribonate, erythronate, and sedoheptulose 7-phosphate in TALDO deficiency. Non-PPP alternations were also noted involving tryptophan, purine, and pyrimidine metabolism for both TKT and TALDO deficient patients. Targeted polyol testing and untargeted metabolomic testing methods were both able to identify specific biochemical patterns indicative of TKT and TALDO deficiency in both plasma and urine samples. In addition, untargeted metabolomics was able to identify novel biomarkers, thereby expanding the current knowledge of both conditions and providing further insight into potential underlying pathophysiological mechanisms. Furthermore, untargeted metabolomic testing offers the advantage of having a single effective biochemical screening test for identification of rare IEMs, like TKT and TALDO deficiencies, that may otherwise go undiagnosed due to their generally non-specific clinical presentations.
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Errores Innatos del Metabolismo de los Carbohidratos/genética , Errores Innatos del Metabolismo/genética , Transaldolasa/deficiencia , Transaldolasa/genética , Transcetolasa/genética , Adulto , Biomarcadores/sangre , Errores Innatos del Metabolismo de los Carbohidratos/sangre , Errores Innatos del Metabolismo de los Carbohidratos/metabolismo , Errores Innatos del Metabolismo de los Carbohidratos/patología , Niño , Preescolar , Cromatografía Liquida , Femenino , Humanos , Lactante , Masculino , Espectrometría de Masas , Errores Innatos del Metabolismo/sangre , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/patología , Metabolómica , Vía de Pentosa Fosfato/genética , Transaldolasa/sangre , Transaldolasa/metabolismo , Transcetolasa/sangre , Transcetolasa/deficiencia , Adulto JovenRESUMEN
Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.
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Metabolismo de los Hidratos de Carbono , Trastorno Peroxisomal/genética , Peroxisomas/metabolismo , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Glucosa/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Factor 2 de la Biogénesis del Peroxisoma , Peroxisomas/genética , TranscriptomaRESUMEN
Lysosomal acid lipase deficiency (LAL-D) is a multi-organ autosomal recessive disease caused by mutations in LIPA. We reviewed data from 681 samples (white blood cells [WBC] n = 625, fibroblasts = 30, liver = 4, amniocytes = 13, chorionic villus = 9) received for analysis of lysosomal acid lipase (LAL) activity over a 15-year period. LIPA sequencing was performed in 49 patients with reduced (n = 26) or deficient (n = 23) LAL activity. The Exome Aggregation Consortium and Genome Aggregation Database dataset were used for LAL-D prevalence calculations. LAL WBC activity was reduced in 67 patients (10.72%) and deficient in 37 (5.92%). The average of LAL activity ± margin of error (CI 95%) was 19.32 ± 0.86 pmol/min/mg for reduced activity patients and 5.90 ± 1.42 pmol/min/mg for deficient patients. The average age at diagnosis for LAL-D was 23.6 years with several patients older than age 30. The correlation between the age at diagnosis and LAL activity showed a significant moderate direct correlation (Pearson's r = 0.46, P < 0.005). Homozygous or compound heterozygous mutations were identified in 9 out of 23 patients with deficient results (detection rate 39.1%). The average LAL activity in molecularly confirmed patients was 4.02 ± 2.02 pmol/min/mg protein, while in molecularly negative patients was 13.886 ± 1.49 pmol/min/mg (P < 0.0001). Twenty-two different mutations were identified including two novel variants (c.309C>A and c.856G>C). A carrier frequency of approximately 1 in 350 was inferred. LAL activity in WBC is a validated tool for LAL-D diagnosis. Higher residual enzymatic activity might result in a milder phenotype leading to diagnosis delay. A cut-off below 12 pmol/min/mg protein might be useful to discriminate patients with LIPA mutations.
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Hígado/patología , Esterol Esterasa/metabolismo , Enfermedad de Wolman/diagnóstico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Fibroblastos/metabolismo , Genotipo , Humanos , Lactante , Recién Nacido , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Estudios Retrospectivos , Esterol Esterasa/genética , Estados Unidos/epidemiología , Enfermedad de Wolman/epidemiología , Enfermedad de Wolman/genética , Adulto Joven , Enfermedad de WolmanRESUMEN
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.
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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
Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration--defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.
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Proteínas de Drosophila/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Células Fotorreceptoras de Invertebrados/efectos de la radiación , Enfermedades de la Retina/genética , Adenosina Trifosfato/biosíntesis , Animales , Citrato (si)-Sintasa/genética , Drosophila , Proteínas de Drosophila/metabolismo , Electrorretinografía , Endocitosis , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Estrés Oxidativo , Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rodopsina/metabolismo , Visión OcularRESUMEN
OBJECTIVE: To interrogate the metabolic profile of five subjects from three families with rare, nonsense and missense mutations in SLC13A5 and Early Infantile Epileptic Encephalopathies (EIEE) characterized by severe, neonatal onset seizures, psychomotor retardation and global developmental delay. METHODS: Mass spectrometry of plasma, CSF and urine was used to identify consistently dysregulated analytes in our subjects. RESULTS: Distinctive elevations of citrate and dysregulation of citric acid cycle intermediates, supporting the hypothesis that loss of SLC13A5 function alters tricarboxylic acid cycle (TCA) metabolism and may disrupt metabolic compartmentation in the brain. SIGNIFICANCE: Our results indicate that analysis of plasma citrate and other TCA analytes in SLC13A5 deficient patients define a diagnostic metabolic signature that can aid in diagnosing children with this disease.
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Ciclo del Ácido Cítrico , Espasmos Infantiles/metabolismo , Simportadores/deficiencia , Simportadores/genética , Niño , Ácido Cítrico/sangre , Femenino , Humanos , Recién Nacido , Masculino , Espectrometría de Masas , Metaboloma , Metabolómica/métodos , Mutación , Mutación Missense , Convulsiones/metabolismo , Espasmos Infantiles/diagnóstico , Secuenciación del ExomaRESUMEN
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.
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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 JovenRESUMEN
Pyrroline-5-carboxylate reductase 2, encoded by PYCR2, is one of the three homologous enzymes that catalyze the last step of proline synthesis. Homozygous variants in PYCR2 have been reported in patients from multiple consanguineous families with hypomyelinating leukodystrophy 10 (HLD10) (MIM: 616420). Here, we report five additional patients from three families with homozygous nonsense or missense variants in PYCR2, identified through clinical exome sequencing. All patients presented with postnatally acquired microcephaly, moderate to profound global developmental delay, and failure to thrive. Brain MRI in these patients showed thin corpus callosum, delayed myelination, and generalized white-matter volume loss. Additional phenotypes that were less consistent among patients included seizures or seizure-like movements, spasticity and ataxic gait, recurrent vomiting, cortical blindness, dysmorphic features, joint contractures, and irritability. Exome sequencing identified homozygous variants in PYCR2 in the proband from each family: c.28C>T (p.(Glu10Ter)), c.796C>T (p.(Arg266Ter)), and c.577G>A (p.(Val193Met)). Subsequent targeted analyses demonstrated co-segregation of the disease with the variant in the family. Despite the metabolic role of PYCR2, routine serum metabolic test in these patients were normal. To further understand the disease etiology and functions of PYCR2, small molecule metabolomics profiling was performed in plasma from three severely affected patients. No significant changes were identified in proline biosynthesis pathway or related metabolites. Studying the clinical features and the metabolic profiles of the PYCR2-deficient patients provides a more comprehensive picture for this newly identified disorder and facilitates further research on the gene function and disease etiology. © 2016 Wiley Periodicals, Inc.
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Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Homocigoto , Microcefalia/diagnóstico , Microcefalia/genética , Mutación , Pirrolina Carboxilato Reductasas/genética , Adolescente , Alelos , Sustitución de Aminoácidos , Encéfalo/anomalías , Niño , Preescolar , Codón , Análisis Mutacional de ADN , Exoma , Femenino , Estudios de Asociación Genética , Gráficos de Crecimiento , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Metabolómica/métodos , Linaje , FenotipoRESUMEN
Mitochondrial dysfunction plays a central role in the neuropathology associated with status epilepticus (SE) and is implicated in the development of epilepsy. While excitotoxic mechanisms are well-known mediators affecting mitochondrial health following SE, whether hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1) also contributes to SE-induced mitochondrial dysfunction remains to be examined. Here we first evaluated the temporal evolution of poly-ADP-ribosylated protein levels in hippocampus following kainic acid-induced SE as a marker for PARP-1 activity, and found that PARP-1 was hyperactive at 24 h following SE. We evaluated oxidative metabolism and found decreased NAD⺠levels by enzymatic cycling, and impaired NADâº-dependent mitochondrial respiration as measured by polarography at 24 h following SE. Stereological estimation showed significant cell loss in the hippocampal CA1 and CA3 subregions 72 h following SE. PARP-1 inhibition using N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)- N,N-dimethylacetamide (PJ-34) in vivo administration was associated with preserved NAD⺠levels and NADâº-dependent mitochondrial respiration, and improved CA1 neuronal survival. These findings suggest that PARP-1 hyperactivation contributes to SE-associated mitochondrial dysfunction and CA1 hippocampal damage. The deleterious effects of PARP-1 hyperactivation on mitochondrial respiration are in part mediated through intracellular NAD⺠depletion. Therefore, modulating PARP-1 activity may represent a potential therapeutic target to preserve intracellular energetics and mitochondrial function following SE.
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Hipocampo/metabolismo , Hipocampo/patología , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas/metabolismo , Neuronas/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Animales , Western Blotting , Electroencefalografía , Ratas , Ratas Sprague-DawleyRESUMEN
Nuclear genetic disorders causing mitochondrial DNA (mtDNA) depletion are clinically and genetically heterogeneous, and the molecular etiology remains undiagnosed in the majority of cases. Through whole-exome sequencing, we identified recessive nonsense and splicing mutations in FBXL4 segregating in three unrelated consanguineous kindreds in which affected children present with a fatal encephalopathy, lactic acidosis, and severe mtDNA depletion in muscle. We show that FBXL4 is an F-box protein that colocalizes with mitochondria and that loss-of-function and splice mutations in this protein result in a severe respiratory chain deficiency, loss of mitochondrial membrane potential, and a disturbance of the dynamic mitochondrial network and nucleoid distribution in fibroblasts from affected individuals. Expression of the wild-type FBXL4 transcript in cell lines from two subjects fully rescued the levels of mtDNA copy number, leading to a correction of the mitochondrial biochemical deficit. Together our data demonstrate that mutations in FBXL4 are disease causing and establish FBXL4 as a mitochondrial protein with a possible role in maintaining mtDNA integrity and stability.
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ADN Mitocondrial/genética , Proteínas F-Box/genética , Predisposición Genética a la Enfermedad , Encefalomiopatías Mitocondriales/genética , Mutación/genética , Ubiquitina-Proteína Ligasas/genética , Acidosis Láctica/complicaciones , Acidosis Láctica/genética , Acidosis Láctica/patología , Secuencia de Bases , Niño , Preescolar , Segregación Cromosómica/genética , Transporte de Electrón/genética , Proteínas F-Box/química , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Dosificación de Gen/genética , Genes Recesivos/genética , Humanos , Lactante , Recién Nacido , Masculino , Encefalomiopatías Mitocondriales/complicaciones , Encefalomiopatías Mitocondriales/patología , Datos de Secuencia Molecular , Músculo Esquelético/patología , Fosforilación Oxidativa , Linaje , Transporte de Proteínas , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Deficiency of the TCA cycle enzyme Succinyl-CoA Synthetase/Ligase (SCS), due to pathogenic variants in subunits encoded by SUCLG1 and SUCLA2, causes mitochondrial encephalomyopathy, methylmalonic acidemia, and mitochondrial DNA (mtDNA) depletion. In this study, we report an 11year old patient who presented with truncal ataxia, chorea, hypotonia, bilateral sensorineural hearing loss and preserved cognition. Whole exome sequencing identified a heterozygous known pathogenic variant and a heterozygous novel missense variant of uncertain clinical significance (VUS) in SUCLG1. To validate the suspected pathogenicity of the novel VUS, molecular and biochemical analyses were performed using primary skin fibroblasts from the patient. The patient's cells lack the SUCLG1 protein, with significantly reduced levels of SUCLA2 and SUCLG2 protein. This leads to essentially undetectable SCS enzyme activity, mtDNA depletion, and cellular respiration defects. These abnormal phenotypes are rescued upon ectopic expression of wild-type SUCLG1 in the patient's fibroblasts, thus functionally confirming the pathogenic nature of the SUCLG1 VUS identified in this patient and expanding the phenotypic spectrum for SUCLG1 deficiency.
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Errores Innatos del Metabolismo de los Aminoácidos/genética , Succinato-CoA Ligasas/genética , Acilcoenzima A/genética , Acilcoenzima A/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/patología , Niño , ADN Mitocondrial/genética , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Heterocigoto , Humanos , Mitocondrias/genética , Mitocondrias/metabolismo , Mutación Missense , Succinato-CoA Ligasas/metabolismo , Secuenciación del ExomaRESUMEN
Defects in the tricarboxylic acid cycle (TCA) are associated with a spectrum of neurological phenotypes that are often difficult to diagnose and manage. Whole-exome sequencing (WES) led to a rapid expansion of diagnostic capabilities in such disorders and facilitated a better understanding of disease pathogenesis, although functional characterization remains a bottleneck to the interpretation of potential pathological variants. We report a 2-year-old boy of Afro-Caribbean ancestry, who presented with neuromuscular symptoms without significant abnormalities on routine diagnostic evaluation. WES revealed compound heterozygous missense variants of uncertain significance in mitochondrial aconitase (ACO2), which encodes the TCA enzyme ACO2. Pathogenic variants in ACO2 have been described in a handful of families as the cause of infantile cerebellar-retinal degeneration syndrome. Using biochemical and cellular assays in patient fibroblasts, we found that ACO2 expression was quantitatively normal, but ACO2 enzyme activity was <20% of that observed in control cells. We also observed a deficiency in cellular respiration and, for the first time, demonstrate evidence of mitochondrial DNA depletion and altered expression of some TCA components and electron transport chain subunits. The observed cellular defects were completely restored with ACO2 gene rescue. Our findings demonstrate the pathogenicity of two VUS in ACO2, provide novel mechanistic insights to TCA disturbances in ACO2 deficiency, and implicate mitochondrial DNA depletion in the pathogenesis of this recently described disorder.
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Aconitato Hidratasa/deficiencia , Aconitato Hidratasa/genética , Errores Innatos del Metabolismo/genética , Mutación Missense , Enfermedades Neuromusculares/genética , Preescolar , Ciclo del Ácido Cítrico , ADN Mitocondrial/genética , Exoma , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Masculino , Errores Innatos del Metabolismo/etnología , Errores Innatos del Metabolismo/metabolismo , Enfermedades Neuromusculares/etnología , Enfermedades Neuromusculares/metabolismoRESUMEN
Aromatic L-amino acid decarboxylase (AADC) deficiency is an inborn error of metabolism affecting the biosynthesis of serotonin, dopamine, and catecholamines. We report a case of AADC deficiency that was detected using the Global MAPS platform. This is a novel platform that allows for parallel clinical testing of hundreds of metabolites in a single plasma specimen. It uses a state-of-the-art mass spectrometry platform, and the resulting spectra are compared against a library of ~2500 metabolites. Our patient is now a 4 year old boy initially seen at 11 months of age for developmental delay and hypotonia. Multiple tests had not yielded a diagnosis until exome sequencing revealed compound heterozygous variants of uncertain significance (VUS), c.286G>A (p.G96R) and c.260C>T (p.P87L) in the DDC gene, causal for AADC deficiency. CSF neurotransmitter analysis confirmed the diagnosis with elevated 3-methoxytyrosine (3-O-methyldopa). Metabolomic profiling was performed on plasma and revealed marked elevation in 3-methoxytyrosine (Z-score +6.1) consistent with the diagnosis of AADC deficiency. These results demonstrate that the Global MAPS platform is able to diagnose AADC deficiency from plasma. In summary, we report a novel and less invasive approach to diagnose AADC deficiency using plasma metabolomic profiling.
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Errores Innatos del Metabolismo de los Aminoácidos/sangre , Descarboxilasas de Aminoácido-L-Aromático/deficiencia , Dopa-Decarboxilasa/genética , Metabolómica/métodos , Polimorfismo de Nucleótido Simple , Descarboxilasas de Aminoácido-L-Aromático/sangre , Dihidroxifenilalanina/análogos & derivados , Dihidroxifenilalanina/sangre , Humanos , Lactante , Masculino , Tirosina/análogos & derivados , Tirosina/sangreRESUMEN
An increasing number of genes required for mitochondrial biogenesis, dynamics, or function have been found to be mutated in metabolic disorders and neurological diseases such as Leigh Syndrome. In a forward genetic screen to identify genes required for neuronal function and survival in Drosophila photoreceptor neurons, we have identified mutations in the mitochondrial methionyl-tRNA synthetase, Aats-met, the homologue of human MARS2. The fly mutants exhibit age-dependent degeneration of photoreceptors, shortened lifespan, and reduced cell proliferation in epithelial tissues. We further observed that these mutants display defects in oxidative phosphorylation, increased Reactive Oxygen Species (ROS), and an upregulated mitochondrial Unfolded Protein Response. With the aid of this knowledge, we identified MARS2 to be mutated in Autosomal Recessive Spastic Ataxia with Leukoencephalopathy (ARSAL) patients. We uncovered complex rearrangements in the MARS2 gene in all ARSAL patients. Analysis of patient cells revealed decreased levels of MARS2 protein and a reduced rate of mitochondrial protein synthesis. Patient cells also exhibited reduced Complex I activity, increased ROS, and a slower cell proliferation rate, similar to Drosophila Aats-met mutants.
Asunto(s)
Ataxia/genética , Proteínas de Drosophila/genética , Drosophila/fisiología , Metionina-ARNt Ligasa/genética , Mitocondrias/enzimología , Enfermedades Neurodegenerativas/genética , Adolescente , Adulto , Animales , Ataxia/metabolismo , Proliferación Celular , Niño , Preescolar , Drosophila/enzimología , Drosophila/genética , Proteínas de Drosophila/metabolismo , Transporte de Electrón , Electrorretinografía/métodos , Femenino , Regulación Enzimológica de la Expresión Génica , Células HEK293 , Humanos , Leucoencefalopatías/genética , Leucoencefalopatías/metabolismo , Longevidad , Masculino , Metionina-ARNt Ligasa/metabolismo , Persona de Mediana Edad , Mitocondrias/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Músculos/metabolismo , Músculos/fisiopatología , Mutación , Enfermedades Neurodegenerativas/metabolismo , Fosforilación Oxidativa , Linaje , Fenotipo , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/patología , Especies Reactivas de Oxígeno/metabolismo , Retina/metabolismo , Retina/patología , Respuesta de Proteína Desplegada , Adulto JovenRESUMEN
Aberrant glucose metabolism characterized by high levels of glycolysis, even in the presence of oxygen, is an important hallmark of cancer. This metabolic reprogramming referred to as the Warburg effect is essential to the survival of tumor cells and provides them with substrates required for biomass generation. Molecular mechanisms responsible for this shift in glucose metabolism remain elusive. As described herein, we found that aberrant expression of the proinflammatory protein transglutaminase 2 (TG2) is an important regulator of the Warburg effect in mammary epithelial cells. Mechanistically, TG2 regulated metabolic reprogramming by constitutively activating nuclear factor (NF)-κB, which binds to the hypoxia-inducible factor (HIF)-1α promoter and induces its expression even under normoxic conditions. TG2/NF-κB-induced increase in HIF-1α expression was associated with increased glucose uptake, increased lactate production and decreased oxygen consumption by mitochondria. Experimental suppression of TG2 attenuated HIF-1α expression and reversed downstream events in mammary epithelial cells. Moreover, downregulation of p65/RelA or HIF-1α expression in these cells restored normal glucose uptake, lactate production, mitochondrial respiration and glycolytic protein expression. Our results suggest that aberrant expression of TG2 is a master regulator of metabolic reprogramming and facilitates metabolic alterations in epithelial cells even under normoxic conditions. A TG2-induced shift in glucose metabolism helps breast cancer cells to survive under stressful conditions and promotes their metastatic competence.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Glucosa/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Glándulas Mamarias Humanas/metabolismo , Factor de Transcripción ReIA/metabolismo , Transglutaminasas/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Respiración de la Célula/genética , Regulación hacia Abajo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/genética , Regulación Neoplásica de la Expresión Génica , Glucólisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Inflamación/inmunología , Ácido Láctico/biosíntesis , Células MCF-7 , Glándulas Mamarias Humanas/patología , Mitocondrias/metabolismo , Oxígeno/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Glutamina Gamma Glutamiltransferasa 2 , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/inmunología , Factor de Transcripción ReIA/biosíntesis , Transglutaminasas/biosíntesis , Transglutaminasas/genética , Regulación hacia ArribaRESUMEN
Mitochondrial myopathy, lactic acidosis and sideroblastic anemia (MLASA) is a rare mitochondrial disorder that has previously been associated with mutations in PUS1 and YARS2. In the present report, we describe a 6-year old male with an MLASA plus phenotype. This patient had features of MLASA in the setting of developmental delay, sensorineural hearing loss, epilepsy, agenesis of the corpus callosum, failure to thrive, and stroke-like episodes. Sequencing of the mitochondrial genome identified a novel de novo, heteroplasmic mutation in the mitochondrial DNA (mtDNA) encoded ATP6 gene (m.8969G>A, p.S148N). Whole exome sequencing did not identify mutations or variants in PUS1 or YARS2 or any known nuclear genes that could affect mitochondrial function and explain this phenotype. Studies of fibroblasts derived from the patient revealed a decrease in oligomycin-sensitive respiration, a finding which is consistent with a complex V defect. Thus, this mutation in MT-ATP6 may represent the first mtDNA point mutation associated with the MLASA phenotype.
Asunto(s)
Acidosis Láctica/diagnóstico , Anemia Sideroblástica/diagnóstico , ADN Mitocondrial/genética , Miopatías Mitocondriales/diagnóstico , ATPasas de Translocación de Protón Mitocondriales/genética , Acidosis Láctica/genética , Secuencia de Aminoácidos , Anemia Sideroblástica/genética , Respiración de la Célula , Células Cultivadas , Niño , Análisis Mutacional de ADN , Estudios de Asociación Genética , Humanos , Masculino , Miopatías Mitocondriales/genética , Datos de Secuencia Molecular , Mutación PuntualRESUMEN
The Tetrahymena thermophila origin recognition complex (ORC) contains an integral RNA subunit, 26T RNA, which confers specificity to the amplified ribosomal DNA (rDNA) origin by base pairing with an essential cis-acting replication determinant--the type I element. Using a plasmid maintenance assay, we identified a 6.7 kb non-rDNA fragment containing two closely associated replicators, ARS1-A (0.8 kb) and ARS1-B (1.2 kb). Both replicators lack type I elements and hence complementarity to 26T RNA, suggesting that ORC is recruited to these sites by an RNA-independent mechanism. Consistent with this prediction, although ORC associated exclusively with origin sequences in the 21 kb rDNA minichromosome, the interaction between ORC and the non-rDNA ARS1 chromosome changed across the cell cycle. In G(2) phase, ORC bound to all tested sequences in a 60 kb interval spanning ARS1-A/B. Remarkably, ORC and Mcm6 associated with just the ARS1-A replicator in G(1) phase when pre-replicative complexes assemble. We propose that ORC is stochastically deposited onto newly replicated non-rDNA chromosomes and subsequently targeted to preferred initiation sites prior to the next S phase.
Asunto(s)
ADN Ribosómico/metabolismo , Complejo de Reconocimiento del Origen/metabolismo , Origen de Réplica , Tetrahymena thermophila/metabolismo , Animales , Secuencia de Bases , Ciclo Celular , Cromosomas/metabolismo , Biología Computacional , Replicación del ADN , ADN Ribosómico/genética , Modelos Biológicos , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Complejo de Reconocimiento del Origen/genética , Unión Proteica , Origen de Réplica/genética , Eliminación de Secuencia , Tetrahymena thermophila/citologíaRESUMEN
Ghrelin receptor, a growth hormone secretagogue receptor (GHS-R), is expressed in the pancreas. Emerging evidence indicates that GHS-R is involved in the regulation of glucose-stimulated insulin secretion (GSIS), but the mechanism by which GHS-R regulates GSIS in the pancreas is unclear. In this study, we investigated the role of GHS-R on GSIS in detail using global Ghsr-/- mice (in vivo) and Ghsr-ablated pancreatic islets (ex vivo). GSIS was attenuated in both Ghsr-/- mice and Ghsr-ablated islets, while the islet morphology was similar between WT and Ghsr-/- mice. To elucidate the mechanism underpinning Ghsr-mediated GSIS, we investigated the key steps of the GSIS signaling cascade. The gene expression of glucose transporter 2 (Glut2) and the glucose-metabolic intermediate-glucose-6-phosphate (G6P) were reduced in Ghsr-ablated islets, supporting decreased glucose uptake. There was no difference in mitochondrial DNA content in the islets of WT and Ghsr-/- mice, but the ATP/ADP ratio in Ghsr-/- islets was significantly lower than that of WT islets. Moreover, the expression of pancreatic and duodenal homeobox 1 (Pdx1), as well as insulin signaling genes of insulin receptor (IR) and insulin receptor substrates 1 and 2 (IRS1/IRS2), was downregulated in Ghsr-/- islets. Akt is the key mediator of the insulin signaling cascade. Concurrently, Akt phosphorylation was reduced in the pancreas of Ghsr-/- mice under both insulin-stimulated and homeostatic conditions. These findings demonstrate that GHS-R ablation affects key components of the insulin signaling pathway in the pancreas, suggesting the existence of a cross-talk between GHS-R and the insulin signaling pathway in pancreatic islets, and GHS-R likely regulates GSIS via the Akt-Pdx1-GLUT2 pathway.