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1.
Brain ; 147(7): 2471-2482, 2024 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-38386308

RESUMEN

Neurodevelopmental disorders are major indications for genetic referral and have been linked to more than 1500 loci including genes encoding transcriptional regulators. The dysfunction of transcription factors often results in characteristic syndromic presentations; however, at least half of these patients lack a genetic diagnosis. The implementation of machine learning approaches has the potential to aid in the identification of new disease genes and delineate associated phenotypes. Next generation sequencing was performed in seven affected individuals with neurodevelopmental delay and dysmorphic features. Clinical characterization included reanalysis of available neuroimaging datasets and 2D portrait image analysis with GestaltMatcher. The functional consequences of ZSCAN10 loss were modelled in mouse embryonic stem cells (mESCs), including a knockout and a representative ZSCAN10 protein truncating variant. These models were characterized by gene expression and western blot analyses, chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) and immunofluorescence staining. Zscan10 knockout mouse embryos were generated and phenotyped. We prioritized bi-allelic ZSCAN10 loss-of-function variants in seven affected individuals from five unrelated families as the underlying molecular cause. RNA-sequencing analyses in Zscan10-/- mESCs indicated dysregulation of genes related to stem cell pluripotency. In addition, we established in mESCs the loss-of-function mechanism for a representative human ZSCAN10 protein truncating variant by showing alteration of its expression levels and subcellular localization, interfering with its binding to DNA enhancer targets. Deep phenotyping revealed global developmental delay, facial asymmetry and malformations of the outer ear as consistent clinical features. Cerebral MRI showed dysplasia of the semicircular canals as an anatomical correlate of sensorineural hearing loss. Facial asymmetry was confirmed as a clinical feature by GestaltMatcher and was recapitulated in the Zscan10 mouse model along with inner and outer ear malformations. Our findings provide evidence of a novel syndromic neurodevelopmental disorder caused by bi-allelic loss-of-function variants in ZSCAN10.


Asunto(s)
Ratones Noqueados , Trastornos del Neurodesarrollo , Adolescente , Animales , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Ratones , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Factores de Transcripción/genética
2.
Am J Hum Genet ; 106(2): 246-255, 2020 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-32004447

RESUMEN

Ral (Ras-like) GTPases play an important role in the control of cell migration and have been implicated in Ras-mediated tumorigenicity. Recently, variants in RALA were also described as a cause of intellectual disability and developmental delay, indicating the relevance of this pathway to neuropediatric diseases. Here, we report the identification of bi-allelic variants in RALGAPA1 (encoding Ral GTPase activating protein catalytic alpha subunit 1) in four unrelated individuals with profound neurodevelopmental disability, muscular hypotonia, feeding abnormalities, recurrent fever episodes, and infantile spasms . Dysplasia of corpus callosum with focal thinning of the posterior part and characteristic facial features appeared to be unifying findings. RalGAPA1 was absent in the fibroblasts derived from two affected individuals suggesting a loss-of-function effect of the RALGAPA1 variants. Consequently, RalA activity was increased in these cell lines, which is in keeping with the idea that RalGAPA1 deficiency causes a constitutive activation of RalA. Additionally, levels of RalGAPB, a scaffolding subunit of the RalGAP complex, were dramatically reduced, indicating a dysfunctional RalGAP complex. Moreover, RalGAPA1 deficiency clearly increased cell-surface levels of lipid raft components in detached fibroblasts, which might indicate that anchorage-dependence of cell growth signaling is disturbed. Our findings indicate that the dysregulation of the RalA pathway has an important impact on neuronal function and brain development. In light of the partially overlapping phenotype between RALA- and RALGAPA1-associated diseases, it appears likely that dysregulation of the RalA signaling pathway leads to a distinct group of genetic syndromes that we suggest could be named RALopathies.


Asunto(s)
Trastornos de Alimentación y de la Ingestión de Alimentos/etiología , Proteínas Activadoras de GTPasa/genética , Hipotonía Muscular/etiología , Mutación , Proteínas del Tejido Nervioso/genética , Trastornos del Neurodesarrollo/etiología , Espasmos Infantiles/etiología , Alelos , Movimiento Celular , Proliferación Celular , Preescolar , Familia , Trastornos de Alimentación y de la Ingestión de Alimentos/patología , Femenino , Humanos , Lactante , Masculino , Hipotonía Muscular/patología , Trastornos del Neurodesarrollo/patología , Fenotipo , Espasmos Infantiles/patología
3.
Genet Med ; 25(7): 100836, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37013901

RESUMEN

PURPOSE: Rothmund-Thomson syndrome (RTS) is characterized by poikiloderma, sparse hair, small stature, skeletal defects, cancer, and cataracts, resembling features of premature aging. RECQL4 and ANAPC1 are the 2 known disease genes associated with RTS in >70% of cases. We describe RTS-like features in 5 individuals with biallelic variants in CRIPT (OMIM 615789). METHODS: Two newly identified and 4 published individuals with CRIPT variants were systematically compared with those with RTS using clinical data, computational analysis of photographs, histologic analysis of skin, and cellular studies on fibroblasts. RESULTS: All CRIPT individuals fulfilled the diagnostic criteria for RTS and additionally had neurodevelopmental delay and seizures. Using computational gestalt analysis, CRIPT individuals showed greatest facial similarity with individuals with RTS. Skin biopsies revealed a high expression of senescence markers (p53/p16/p21) and the senescence-associated ß-galactosidase activity was elevated in CRIPT-deficient fibroblasts. RECQL4- and CRIPT-deficient fibroblasts showed an unremarkable mitotic progression and unremarkable number of mitotic errors and no or only mild sensitivity to genotoxic stress by ionizing radiation, mitomycin C, hydroxyurea, etoposide, and potassium bromate. CONCLUSION: CRIPT causes an RTS-like syndrome associated with neurodevelopmental delay and epilepsy. At the cellular level, RECQL4- and CRIPT-deficient cells display increased senescence, suggesting shared molecular mechanisms leading to the clinical phenotypes.


Asunto(s)
Síndrome Rothmund-Thomson , Humanos , Síndrome Rothmund-Thomson/genética , Síndrome Rothmund-Thomson/diagnóstico , Síndrome Rothmund-Thomson/patología , Senescencia Celular/genética , Daño del ADN , Hidroxiurea/metabolismo , Fibroblastos , Mutación , Proteínas Adaptadoras Transductoras de Señales/metabolismo
4.
Genet Med ; 25(7): 100838, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37057673

RESUMEN

PURPOSE: Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell growth in response to nutritional status. Central to the mTORC1 function is the Rag-GTPase heterodimer. One component of the Rag heterodimer is RagC (Ras-related GTP-binding protein C), which is encoded by the RRAGC gene. METHODS: Genetic testing via trio exome sequencing was applied to identify the underlying disease cause in 3 infants with dilated cardiomyopathy, hepatopathy, and brain abnormalities, including pachygyria, polymicrogyria, and septo-optic dysplasia. Studies in patient-derived skin fibroblasts and in a HEK293 cell model were performed to investigate the cellular consequences. RESULTS: We identified 3 de novo missense variants in RRAGC (NM_022157.4: c.269C>A, p.(Thr90Asn), c.353C>T, p.(Pro118Leu), and c.343T>C, p.(Trp115Arg)), which were previously reported as occurring somatically in follicular lymphoma. Studies of patient-derived fibroblasts carrying the p.(Thr90Asn) variant revealed increased cell size, as well as dysregulation of mTOR-related p70S6K (ribosomal protein S6 kinase 1) and transcription factor EB signaling. Moreover, subcellular localization of mTOR was decoupled from metabolic state. We confirmed the key findings for all RRAGC variants described in this study in a HEK293 cell model. CONCLUSION: The above results are in line with a constitutive overactivation of the mTORC1 pathway. Our study establishes de novo missense variants in RRAGC as cause of an early-onset mTORopathy with unfavorable prognosis.


Asunto(s)
Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de Unión al GTP Monoméricas , Serina-Treonina Quinasas TOR , Humanos , Lactante , Fibroblastos/metabolismo , Enfermedades Genéticas Congénitas/genética , Células HEK293 , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Complejos Multiproteicos/genética , Mutación Missense , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo
5.
J Med Genet ; 59(9): 878-887, 2022 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34656997

RESUMEN

BACKGROUND: Human coenzyme Q4 (COQ4) is essential for coenzyme Q10 (CoQ10) biosynthesis. Pathogenic variants in COQ4 cause childhood-onset neurodegeneration. We aimed to delineate the clinical spectrum and the cellular consequences of COQ4 deficiency. METHODS: Clinical course and neuroradiological findings in a large cohort of paediatric patients with COQ4 deficiency were analysed. Functional studies in patient-derived cell lines were performed. RESULTS: We characterised 44 individuals from 36 families with COQ4 deficiency (16 newly described). A total of 23 different variants were identified, including four novel variants in COQ4. Correlation analyses of clinical and neuroimaging findings revealed three disease patterns: type 1: early-onset phenotype with neonatal brain anomalies and epileptic encephalopathy; type 2: intermediate phenotype with distinct stroke-like lesions; and type 3: moderate phenotype with non-specific brain pathology and a stable disease course. The functional relevance of COQ4 variants was supported by in vitro studies using patient-derived fibroblast lines. Experiments revealed significantly decreased COQ4 protein levels, reduced levels of cellular CoQ10 and elevated levels of the metabolic intermediate 6-demethoxyubiquinone. CONCLUSION: Our study describes the heterogeneous clinical presentation of COQ4 deficiency and identifies phenotypic subtypes. Cell-based studies support the pathogenic characteristics of COQ4 variants. Due to the insufficient clinical response to oral CoQ10 supplementation, alternative treatment strategies are warranted.


Asunto(s)
Proteínas Mitocondriales , Ubiquinona , Línea Celular , Niño , Humanos , Recién Nacido , Proteínas Mitocondriales/genética , Neuroimagen , Fenotipo , Ubiquinona/genética , Ubiquinona/metabolismo
6.
Mov Disord ; 37(10): 2147-2153, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36047608

RESUMEN

BACKGROUND: COQ4 codes for a mitochondrial protein required for coenzyme Q10 (CoQ10 ) biosynthesis. Autosomal recessive COQ4-associated CoQ10 deficiency leads to an early-onset mitochondrial multi-organ disorder. METHODS: In-house exome and genome datasets (n = 14,303) were screened for patients with bi-allelic variants in COQ4. Work-up included clinical characterization and functional studies in patient-derived cell lines. RESULTS: Six different COQ4 variants, three of them novel, were identified in six adult patients from four different families. Three patients had a phenotype of hereditary spastic paraparesis, two sisters showed a predominant cerebellar ataxia, and one patient had mild signs of both. Studies in patient-derived fibroblast lines revealed significantly reduced amounts of COQ4 protein, decreased CoQ10 concentrations, and elevated levels of the metabolic intermediate 6-demethoxyubiquinone. CONCLUSION: We report bi-allelic variants in COQ4 causing an adult-onset ataxia-spasticity spectrum phenotype and a disease course much milder than previously reported. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Asunto(s)
Ataxia Cerebelosa , Proteínas Mitocondriales , Ubiquinona , Ataxia/genética , Ataxia Cerebelosa/genética , Humanos , Enfermedades Mitocondriales , Proteínas Mitocondriales/genética , Espasticidad Muscular , Debilidad Muscular , Mutación/genética , Ubiquinona/deficiencia , Ubiquinona/genética , Ubiquinona/metabolismo
7.
Brain ; 143(1): 94-111, 2020 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-31855247

RESUMEN

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance.


Asunto(s)
Antígenos CD/genética , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Proteínas de Transporte de Catión Orgánico/genética , Adolescente , Ataxia/genética , Ataxia/fisiopatología , Atrofia , Cerebelo/diagnóstico por imagen , Cerebelo/patología , Colina/farmacología , Disfunción Cognitiva/genética , Disfunción Cognitiva/fisiopatología , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/ultraestructura , Trastornos de Deglución/genética , Trastornos de Deglución/fisiopatología , Disartria/genética , Disartria/fisiopatología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/ultraestructura , Incontinencia Fecal/genética , Incontinencia Fecal/fisiopatología , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/ultraestructura , Mutación del Sistema de Lectura , Globo Pálido/diagnóstico por imagen , Trastornos Heredodegenerativos del Sistema Nervioso/diagnóstico por imagen , Trastornos Heredodegenerativos del Sistema Nervioso/patología , Trastornos Heredodegenerativos del Sistema Nervioso/fisiopatología , Homocigoto , Humanos , Leucoencefalopatías/diagnóstico por imagen , Leucoencefalopatías/genética , Leucoencefalopatías/fisiopatología , Imagen por Resonancia Magnética , Masculino , Microscopía Electrónica , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Nootrópicos/farmacología , Atrofia Óptica/genética , Atrofia Óptica/fisiopatología , Linaje , Ribosomas/efectos de los fármacos , Ribosomas/ultraestructura , Sustancia Negra/diagnóstico por imagen , Síndrome , Temblor/genética , Temblor/fisiopatología , Incontinencia Urinaria/genética , Incontinencia Urinaria/fisiopatología
9.
Brain ; 142(12): 3876-3891, 2019 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-31688942

RESUMEN

Ohtahara syndrome, early infantile epileptic encephalopathy with a suppression burst EEG pattern, is an aetiologically heterogeneous condition starting in the first weeks or months of life with intractable seizures and profound developmental disability. Using whole exome sequencing, we identified biallelic DMXL2 mutations in three sibling pairs with Ohtahara syndrome, belonging to three unrelated families. Siblings in Family 1 were compound heterozygous for the c.5135C>T (p.Ala1712Val) missense substitution and the c.4478C>G (p.Ser1493*) nonsense substitution; in Family 2 were homozygous for the c.4478C>A (p.Ser1493*) nonsense substitution and in Family 3 were homozygous for the c.7518-1G>A (p.Trp2507Argfs*4) substitution. The severe developmental and epileptic encephalopathy manifested from the first day of life and was associated with deafness, mild peripheral polyneuropathy and dysmorphic features. Early brain MRI investigations in the first months of life revealed thin corpus callosum with brain hypomyelination in all. Follow-up MRI scans in three patients revealed progressive moderate brain shrinkage with leukoencephalopathy. Five patients died within the first 9 years of life and none achieved developmental, communicative or motor skills following birth. These clinical findings are consistent with a developmental brain disorder that begins in the prenatal brain, prevents neural connections from reaching the expected stages at birth, and follows a progressive course. DMXL2 is highly expressed in the brain and at synaptic terminals, regulates v-ATPase assembly and activity and participates in intracellular signalling pathways; however, its functional role is far from complete elucidation. Expression analysis in patient-derived skin fibroblasts demonstrated absence of the DMXL2 protein, revealing a loss of function phenotype. Patients' fibroblasts also exhibited an increased LysoTracker® signal associated with decreased endolysosomal markers and degradative processes. Defective endolysosomal homeostasis was accompanied by impaired autophagy, revealed by lower LC3II signal, accumulation of polyubiquitinated proteins, and autophagy receptor p62, with morphological alterations of the autolysosomal structures on electron microscopy. Altered lysosomal homeostasis and defective autophagy were recapitulated in Dmxl2-silenced mouse hippocampal neurons, which exhibited impaired neurite elongation and synaptic loss. Impaired lysosomal function and autophagy caused by biallelic DMXL2 mutations affect neuronal development and synapse formation and result in Ohtahara syndrome with profound developmental impairment and reduced life expectancy.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Autofagia/genética , Encéfalo/fisiopatología , Proteínas del Tejido Nervioso/genética , Espasmos Infantiles/genética , Encéfalo/diagnóstico por imagen , Niño , Preescolar , Progresión de la Enfermedad , Electroencefalografía , Femenino , Humanos , Lactante , Lisosomas/fisiología , Imagen por Resonancia Magnética , Masculino , Mutación , Linaje , Espasmos Infantiles/diagnóstico por imagen , Espasmos Infantiles/fisiopatología , Secuenciación del Exoma
10.
Am J Hum Genet ; 99(4): 894-902, 2016 Oct 06.
Artículo en Inglés | MEDLINE | ID: mdl-27616477

RESUMEN

To safeguard the cell from the accumulation of potentially harmful metabolic intermediates, specific repair mechanisms have evolved. APOA1BP, now renamed NAXE, encodes an epimerase essential in the cellular metabolite repair for NADHX and NADPHX. The enzyme catalyzes the epimerization of NAD(P)HX, thereby avoiding the accumulation of toxic metabolites. The clinical importance of the NAD(P)HX repair system has been unknown. Exome sequencing revealed pathogenic biallelic mutations in NAXE in children from four families with (sub-) acute-onset ataxia, cerebellar edema, spinal myelopathy, and skin lesions. Lactate was elevated in cerebrospinal fluid of all affected individuals. Disease onset was during the second year of life and clinical signs as well as episodes of deterioration were triggered by febrile infections. Disease course was rapidly progressive, leading to coma, global brain atrophy, and finally to death in all affected individuals. NAXE levels were undetectable in fibroblasts from affected individuals of two families. In these fibroblasts we measured highly elevated concentrations of the toxic metabolite cyclic-NADHX, confirming a deficiency of the mitochondrial NAD(P)HX repair system. Finally, NAD or nicotinic acid (vitamin B3) supplementation might have therapeutic implications for this fatal disorder.


Asunto(s)
Proteínas Portadoras/genética , Enfermedades Metabólicas/genética , Mutación , NAD/análogos & derivados , Enfermedades del Sistema Nervioso/genética , Racemasas y Epimerasas/genética , Proteínas Portadoras/metabolismo , Línea Celular , Preescolar , Resultado Fatal , Femenino , Fibroblastos , Humanos , Lactante , Masculino , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/patología , NAD/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Enfermedades del Sistema Nervioso/patología , Neuroimagen , Anomalías Cutáneas/genética , Anomalías Cutáneas/patología
11.
Biochim Biophys Acta Mol Basis Dis ; 1863(6): 1605-1614, 2017 06.
Artículo en Inglés | MEDLINE | ID: mdl-28235645

RESUMEN

Peri-conceptional exposure to maternal obesogenic nutrition is associated with in utero programming of later-life overweight and metabolic disease in the offspring. We aimed to investigate whether dietary intervention with a modified fatty acid quality in an obesogenic high-calorie (HC) diet during the preconception and gestational phases can improve unfavourable effects of an adipogenic maternal environment. In NMRI mice, peri-conceptional and gestational obesity was induced by feeding a HC diet (controls), and they were compared with dams on a fat-modified (Fat-mod) HC diet of the same energy content but enriched with medium-chain fatty acids (MCFAs) and adjusted to a decreased ratio of n-6 to n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). Effects on maternal and placental outcomes at delivery (day 17.5 post coitum) were investigated. Despite comparable energy assimilation between the two groups of dams, the altered fatty acid composition of the Fat-mod HC diet induced lower maternal body weight, weights of fat depots, adipocyte size, and hepatic fat accumulation compared to the unmodified HC diet group. Further, there was a trend towards lower fasting glucose, insulin and leptin concentrations in dams fed the Fat-mod HC diet. Phenotypic changes were accompanied by inhibition of transcript and protein expression of genes involved in hepatic de novo lipogenesis comprising PPARG2 and its target genes Fasn, Acaca, and Fabp4, whereas regulation of other lipogenic factors (Srebf1, Nr1h3, Abca1) appeared to be more complex. The modified diet led to a sex-specific placental response by upregulating PPARG-dependent fatty acid transport gene expression in female versus male placentae. Qualitative modification of the fatty acid spectrum of a high-energy maternal diet, using a combination of both MCFAs and n-3 LC-PUFAs, seems to be a promising interventional approach to ameliorate the adipogenic milieu of mice before and during gestation.


Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Regulación del Desarrollo de la Expresión Génica , Obesidad/metabolismo , Placenta/metabolismo , Complicaciones del Embarazo/metabolismo , Proteínas Gestacionales/biosíntesis , Animales , Femenino , Ratones , Ratones Obesos , Obesidad/inducido químicamente , Obesidad/patología , Placenta/patología , Embarazo , Complicaciones del Embarazo/inducido químicamente , Complicaciones del Embarazo/patología
12.
Mol Genet Metab ; 121(3): 216-223, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28552678

RESUMEN

Coenzyme Q10 (CoQ10) is an essential cofactor of the mitochondrial oxidative phosphorylation (OXPHOS) system and its deficiency has important implications for several inherited metabolic disorders of childhood. The biosynthesis of CoQ10 is a complicated process, which involves at least 12 different enzymes. One of the metabolic intermediates that are formed during CoQ10 biosynthesis is the molecule 6-demethoxyubiquinone (6-DMQ). This CoQ precursor is processed at the level of COQ7 and COQ9. We selected this metabolite as a marker substance for metabolic analysis of cell lines with inherited genetic defects (COQ2, COQ4, COQ7 and COQ9) or siRNA knockdown in CoQ biosynthesis enzymes using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). In COQ4, COQ7 and COQ9 deficient cell lines, we detected significantly elevated levels of 6-DMQ. This suggests a functional interplay of these proteins. However, additional siRNA studies demonstrated that elevated 6-DMQ levels are not an exclusive marker of the COQ7/COQ9 enzymatic step of CoQ10 biosynthesis but constitute a more general phenomenon that occurs in disorders impairing the function or stability of the CoQ-synthome. To further investigate the interdependence of CoQ10 biosynthesis enzyme expression, we performed immunoblotting in various cell lines with CoQ10 deficiency, indicating that COQ4, COQ7 and COQ9 protein expression levels are highly regulated depending on the underlying defect. Supplementation of cell lines with synthetic CoQ precursor compounds demonstrated beneficial effects of 2,4-dihydroxybenzoic acid in COQ7 and COQ9 deficiency. Moreover, vanillic acid selectively stimulated CoQ10 biosynthesis and improved cell viability in COQ9 deficiency. However, compounds tested in this study failed to rescue COQ4 deficiency.


Asunto(s)
Ataxia/metabolismo , Enfermedades Mitocondriales/metabolismo , Debilidad Muscular/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/deficiencia , Línea Celular , Supervivencia Celular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Humanos , Hidroxibenzoatos/farmacología , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Espectrometría de Masas en Tándem , Ubiquinona/biosíntesis , Ubiquinona/metabolismo , Ácido Vanílico/farmacología
14.
Metab Brain Dis ; 32(1): 267-270, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27502409

RESUMEN

VARS2 encodes a mitochondrial aminoacyl-tRNA-synthetase. Mutations in VARS2 have recently been identified as a cause of mitochondrial encephalomyopathy in three individuals. However, clinical information remained scarce. Exome sequencing lead us to identify compound heterozygous pathogenic VARS2 variants in a boy presenting with severe lactic acidosis, hypertrophic cardiomyopathy, epilepsy, and abnormalities on brain imaging including hypoplasia of corpus callosum and cerebellum as well as a massive lactate peak on MR-spectroscopy. Studies in patient-derived fibroblasts confirmed the functional relevance of the identified VARS2 variants. Our report expands the phenotypic spectrum associated with this rare mitochondrial defect, in that VARS2 deficiency may also cause severe neonatal presentations with cardiac involvement and structural brain abnormalities.


Asunto(s)
Cardiomiopatía Hipertrófica/genética , Epilepsia/genética , Antígenos HLA/genética , Encefalomiopatías Mitocondriales/genética , Mutación , Valina-ARNt Ligasa/genética , Análisis Mutacional de ADN , Exoma , Humanos , Masculino
15.
Biochim Biophys Acta ; 1852(3): 529-40, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25536029

RESUMEN

Dysfunction of complex I (CI) of the mitochondrial electron transport chain (ETC) features prominently in human pathology. Cell models of ETC dysfunction display adaptive survival responses that still are poorly understood but of relevance for therapy development. Here we comprehensively examined how primary human skin fibroblasts adapt to chronic CI inhibition. CI inhibition triggered transient and sustained changes in metabolism, redox homeostasis and mitochondrial (ultra)structure but no cell senescence/death. CI-inhibited cells consumed no oxygen and displayed minor mitochondrial depolarization, reverse-mode action of complex V, a slower proliferation rate and futile mitochondrial biogenesis. Adaptation was neither prevented by antioxidants nor associated with increased PGC1-α/SIRT1/mTOR levels. Survival of CI-inhibited cells was strictly glucose-dependent and accompanied by increased AMPK-α phosphorylation, which occurred without changes in ATP or cytosolic calcium levels. Conversely, cells devoid of AMPK-α died upon CI inhibition. Chronic CI inhibition did not increase mitochondrial superoxide levels or cellular lipid peroxidation and was paralleled by a specific increase in SOD2/GR, whereas SOD1/CAT/Gpx1/Gpx2/Gpx5 levels remained unchanged. Upon hormone stimulation, fully adapted cells displayed aberrant cytosolic and ER calcium handling due to hampered ATP fueling of ER calcium pumps. It is concluded that CI dysfunction triggers an adaptive program that depends on extracellular glucose and AMPK-α. This response avoids cell death by suppressing energy crisis, oxidative stress induction and substantial mitochondrial depolarization.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Fibroblastos/enzimología , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismo , Estrés Oxidativo , Transducción de Señal , Proteínas Quinasas Activadas por AMP/genética , Animales , Calcio/metabolismo , Línea Celular Transformada , Supervivencia Celular/genética , Cloruros/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Fibroblastos/citología , Humanos , Ratones , Ratones Noqueados , Mitocondrias/genética , Sirtuina 1/genética , Sirtuina 1/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
16.
Metab Brain Dis ; 31(3): 717-21, 2016 06.
Artículo en Inglés | MEDLINE | ID: mdl-26780086

RESUMEN

Mitochondrial aminoacyl tRNA synthetases are essential for organelle protein synthesis. Genetic defects affecting the function of these enzymes may cause pediatric mitochondrial disease. Here, we report on a child with fatal neonatal lactic acidosis and recurrent hypoglycemia caused by mutations in EARS2, encoding mitochondrial glutamyl-tRNA synthetase 2. Brain ultrasound revealed agenesis of corpus callosum. Studies on patient-derived skin fibroblasts showed severely decreased EARS2 protein levels, elevated reactive oxygen species (ROS) production, and altered mitochondrial morphology. Our report further illustrates the clinical spectrum of the severe neonatal-onset form of EARS2 mutations. Moreover, in this case the live-cell parameters appeared to be more sensitive to mitochondrial dysfunction compared to standard diagnostics, which indicates the potential relevance of fibroblast studies in children with mitochondrial diseases.


Asunto(s)
Acidosis Láctica/genética , Agenesia del Cuerpo Calloso/genética , Glutamato-ARNt Ligasa/genética , Hipoglucemia/genética , Enfermedades Mitocondriales/genética , Acidosis Láctica/diagnóstico por imagen , Agenesia del Cuerpo Calloso/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Resultado Fatal , Humanos , Hipoglucemia/diagnóstico por imagen , Recién Nacido , Masculino , Enfermedades Mitocondriales/diagnóstico por imagen , Recurrencia , Ultrasonografía
17.
Neurogenetics ; 16(3): 237-40, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25663021

RESUMEN

The mitochondrial ribosomes are required for the synthesis of mitochondrial DNA-encoded subunits of the oxidative phosphorylation (OXPHOS) system. Here, we present a neonate with fatal lactic acidosis and combined OXPHOS deficiency caused by a homozygous mutation in MRPS22, a gene encoding a mitochondrial ribosomal small subunit protein. Brain imaging revealed several structural abnormalities, including agenesis of the corpus callosum, multiple periventricular cysts, and suspected intracerebral calcifications. Moreover, echocardiography demonstrated atrial and ventricular septal defects as well as a coronary artery fistula. Our report expands the clinical spectrum of this rare mitochondrial disorder and confirms the severe clinical phenotype associated with this defect.


Asunto(s)
Acidosis Láctica/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Proteínas Ribosómicas/genética , Acidosis Láctica/complicaciones , Encéfalo/patología , Resultado Fatal , Fibroblastos/metabolismo , Mutación del Sistema de Lectura , Humanos , Recién Nacido , Masculino , Enfermedades Mitocondriales/complicaciones , Proteínas Mitocondriales/metabolismo , Miocardio/patología , Proteínas Ribosómicas/metabolismo
18.
Stem Cell Res Ther ; 13(1): 256, 2022 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-35715829

RESUMEN

The most frequent biochemical defect of inherited mitochondrial disease is isolated complex I deficiency. There is no cure for this disorder, and treatment is mainly supportive. In this study, we investigated the effects of human mesenchymal stem cells (MSCs) on skin fibroblast derived from three individuals with complex I deficiency carrying different pathogenic variants in mitochondrial DNA-encoded subunits (MT-ND3, MT-ND6). Complex I-deficient fibroblasts were transiently co-cultured with bone marrow-derived MSCs. Mitochondrial transfer was analysed by fluorescence labelling and validated by Sanger sequencing. Levels of reactive oxygen species (ROS) were measured using MitoSOX Red. Moreover, mitochondrial respiration was analysed by Seahorse XFe96 Extracellular Flux Analyzer. Levels of antioxidant proteins were investigated via immunoblotting. Co-culturing of complex I-deficient fibroblast with MSCs lowered cellular ROS levels. The effect on ROS production was more sustained compared to treatment of patient fibroblasts with culture medium derived from MSC cultures. Investigation of cellular antioxidant defence systems revealed an upregulation of SOD2 (superoxide dismutase 2, mitochondrial) and HO-1 (heme oxygenase 1) in patient-derived cell lines. This adaptive response was normalised upon MSC treatment. Moreover, Seahorse experiments revealed a significant improvement of mitochondrial respiration, indicating a mitigation of the oxidative phosphorylation defect. Experiments with repetitive MSC co-culture at two consecutive time points enhanced this effect. Our study indicates that MSC-based treatment approaches might constitute an interesting option for patients with mitochondrial DNA-encoded mitochondrial diseases. We suggest that this strategy may prove more promising for defects caused by mitochondrial DNA variants compared to nuclear-encoded defects.


Asunto(s)
Antioxidantes , Células Madre Mesenquimatosas , Antioxidantes/metabolismo , Línea Celular , ADN Mitocondrial/genética , Complejo I de Transporte de Electrón/deficiencia , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Fibroblastos/metabolismo , Homeostasis , Humanos , Células Madre Mesenquimatosas/metabolismo , Enfermedades Mitocondriales , NADH Deshidrogenasa/metabolismo , Oxidación-Reducción , Especies Reactivas de Oxígeno/metabolismo , Respiración
19.
J Neuroinflammation ; 8: 51, 2011 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-21592385

RESUMEN

BACKGROUND: A critical point during the course of bacterial meningitis is the excessive influx of polymorphnuclear neutrophils (PMNs) from the blood into the brain. Both paracellular and transcellular routes of leukocyte transmigration through the blood-brain barrier have been described in CNS diseases so far. Thus, we investigated the mechanism of PMN transmigration through the blood-CSF barrier under inflammatory conditions. METHODS: In an "inverted" Transwell culture model of the blood-CSF barrier, the zoonotic agent Streptococcus suis (S. suis) was used to stimulate porcine choroid plexus epithelial cells (PCPECs) specifically from the physiologically relevant basolateral side. Barrier function was analyzed by measuring TEER and TR-dextran-flux, and tight junction morphology was investigated by immunofluorescence. Route and mechanism of PMN transmigration were determined by immunofluorescence, electron microscopy and FACS analysis. Quantitative real time-PCR was used to determine expression levels of ICAM-1 and VCAM-1. RESULTS: Here, we show that the transmigration of PMNs through PCPECs was significantly higher after stimulation with TNFα or infection with S. suis strain 10 compared to its non-encapsulated mutant. Barrier function was not significantly affected by PMN migration alone, but in combination with S. suis infection. Tight junction and cytoskeletal actin reorganisation were also observed after stimulation with S. suis or TNFα. Most strikingly, PMNs preferentially migrated across PCPECs via the transcellular route. Extensive sequential analyses of the PMN transmigration process with Apotome(®)-imaging and electron microscopy revealed that paracellular migrating PMNs stop just before tight junctions. Interestingly, PMNs subsequently appeared to proceed by transcellular migration via funnel-like structures developing from the apical membrane. It is noteworthy that some PMNs contained bacteria during the transmigration process. Flow cytometric and transmigration inhibition studies with integrin-specific antibodies showed that PMN traversal is dependent on CD11b/CD18. Analysis of cell adhesion molecules in PCPECs revealed a significant increase of ICAM-1 and VCAM-1 expression after TNFα and S. suis stimulation. CONCLUSION: Our data underline the relevance of the blood-CSF barrier as a gate for leukocyte entry into the CNS and suggest a novel transcellular migration step during the pathogenesis of bacterial meningitis.


Asunto(s)
Barrera Hematoencefálica/fisiología , Neutrófilos/fisiología , Streptococcus suis/patogenicidad , Migración Transcelular de la Célula/fisiología , Actinas/metabolismo , Actinas/ultraestructura , Animales , Barrera Hematoencefálica/citología , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Células Cultivadas , Plexo Coroideo/citología , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Impedancia Eléctrica , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Humanos , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Neutrófilos/ultraestructura , Porcinos , Uniones Estrechas/metabolismo , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo
20.
Cell Death Dis ; 12(10): 885, 2021 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-34584066

RESUMEN

Glioblastoma is the most common malignant primary brain tumor. To date, clinically relevant biomarkers are restricted to isocitrate dehydrogenase (IDH) gene 1 or 2 mutations and O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Long non-coding RNAs (lncRNAs) have been shown to contribute to glioblastoma pathogenesis and could potentially serve as novel biomarkers. The clinical significance of HOXA Transcript Antisense RNA, Myeloid-Specific 1 (HOTAIRM1) was determined by analyzing HOTAIRM1 in multiple glioblastoma gene expression data sets for associations with prognosis, as well as, IDH mutation and MGMT promoter methylation status. Finally, the role of HOTAIRM1 in glioblastoma biology and radiotherapy resistance was characterized in vitro and in vivo. We identified HOTAIRM1 as a candidate lncRNA whose up-regulation is significantly associated with shorter survival of glioblastoma patients, independent from IDH mutation and MGMT promoter methylation. Glioblastoma cell line models uniformly showed reduced cell viability, decreased invasive growth and diminished colony formation capacity upon HOTAIRM1 down-regulation. Integrated proteogenomic analyses revealed impaired mitochondrial function and determination of reactive oxygen species (ROS) levels confirmed increased ROS levels upon HOTAIRM1 knock-down. HOTAIRM1 knock-down decreased expression of transglutaminase 2 (TGM2), a candidate protein implicated in mitochondrial function, and knock-down of TGM2 mimicked the phenotype of HOTAIRM1 down-regulation in glioblastoma cells. Moreover, HOTAIRM1 modulates radiosensitivity of glioblastoma cells both in vitro and in vivo. Our data support a role for HOTAIRM1 as a driver of biological aggressiveness, radioresistance and poor outcome in glioblastoma. Targeting HOTAIRM1 may be a promising new therapeutic approach.


Asunto(s)
Glioblastoma/genética , Glioblastoma/radioterapia , MicroARNs/metabolismo , Tolerancia a Radiación/genética , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Células Clonales , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/patología , Humanos , Ratones Desnudos , MicroARNs/genética , Mitocondrias/metabolismo , Invasividad Neoplásica , Fenotipo , Pronóstico , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Proteogenómica , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo
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