RESUMO
Given that oropharyngeal squamous cell carcinoma (OPSCC) have now surpassed cervical cancer as the most common human papillomavirus (HPV)-driven cancer, there is an interest in developing non-invasive predictive biomarkers to early detect HPV-driven OPSCC. In total, 665 cancer-free individuals were recruited from Queensland, Australia. Oral HPV16 DNA positivity in those individuals was determined by our in-house developed sensitive PCR method. Individuals with (n = 9) or without (n = 12) oral HPV16 infections at baseline were followed for a median duration of 24 mo. Individuals with persistent oral HPV16 infection (≥ 30 mo) were invited for clinical examination of their oral cavity and oropharynx by an otolaryngologist. Oral HPV16 DNA was detected in 12 out of 650 cancer-free individuals (1.8%; 95% confidence interval [CI]: 1.0-3.2). Of the 3 individuals with persistent oral HPV16 infection, the first individual showed no clinical evidence of pathology. The second individual was diagnosed with a 2 mm invasive squamous cell carcinoma (T1N0M0) positive for both p16INK4a expression and HPV16 DNA. The third individual was found to have a mildly dysplastic lesion in the tonsillar region that was negative for p16INK4a expression and HPV16 DNA and she continues to have HPV16 DNA in her saliva. Taken together, our data support the value of using an oral HPV16 DNA assay as a potential screening tool for the detection of microscopic HPV-driven OPSCC. Larger multicenter studies across various geographic regions recruiting populations at a higher risk of developing HPV-driven OPSCC are warranted to extend and confirm the results of the current investigation.
Assuntos
DNA Viral/isolamento & purificação , Detecção Precoce de Câncer , Papillomavirus Humano 16/patogenicidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Saliva/virologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Adulto JovemRESUMO
OBJECTIVE: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 function causes MLC. MLC1 is involved in ion-water homeostasis, but its exact role is unknown. We generated Mlc1-null mice for further studies. METHODS: We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1-null mice. RESULTS: Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid-brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1-null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid-brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1-null astrocytes showed slowed regulatory volume decrease and reduced volume-regulated anion currents, which increased upon MLC1 re-expression. Mlc1-null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC-2, but no substantial changes in other known MLC1-interacting proteins. INTERPRETATION: Mlc1-null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC-2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC.
Assuntos
Cistos/genética , Cistos/patologia , Cistos/fisiopatologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Adolescente , Adulto , Fatores Etários , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Astrócitos/patologia , Edema Encefálico/etiologia , Cerebelo/patologia , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Criança , Pré-Escolar , Cistos/metabolismo , Modelos Animais de Doenças , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Lactente , Recém-Nascido , Potenciais da Membrana/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/genética , Equilíbrio Postural/genética , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Transtornos de Sensação/genética , Substância Branca/metabolismo , Substância Branca/patologia , Substância Branca/ultraestrutura , Adulto JovemRESUMO
Leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation (LBSL) has recently been defined based on a highly characteristic constellation of abnormalities observed by magnetic resonance imaging and spectroscopy. LBSL is an autosomal recessive disease, most often manifesting in early childhood. Affected individuals develop slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction, sometimes with a mild cognitive deficit or decline. We performed linkage mapping with microsatellite markers in LBSL families and found a candidate region on chromosome 1, which we narrowed by means of shared haplotypes. Sequencing of genes in this candidate region uncovered mutations in DARS2, which encodes mitochondrial aspartyl-tRNA synthetase, in affected individuals from all 30 families. Enzyme activities of mutant proteins were decreased. We were surprised to find that activities of mitochondrial complexes from fibroblasts and lymphoblasts derived from affected individuals were normal, as determined by different assays.
Assuntos
Aspartato-tRNA Ligase/genética , Ligação Genética , Ácido Láctico/metabolismo , Mitocôndrias/genética , Degenerações Espinocerebelares/genética , Aspartato-tRNA Ligase/metabolismo , Marcadores Genéticos , Haplótipos , Humanos , Mitocôndrias/enzimologia , Doenças Mitocondriais/genética , Polimorfismo Genético , Degenerações Espinocerebelares/metabolismoRESUMO
Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is a disorder caused by recessive mutations in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. Recent observations indicate that the phenotypic range of the disease is much wider than initially thought. Currently, no treatment is available. The aims of our study were (i) to explore a possible genotype-phenotype correlation; and (ii) to identify potential therapeutic agents that modulate the splice site mutations in intron 2 of DARS2, present in almost all patients. A cross-sectional observational study was performed in 78 patients with two DARS2 mutations in the Amsterdam and Helsinki databases up to December 2012. Clinical information was collected via questionnaires. An inventory was made of the DARS2 mutations in these patients and those previously published. An assay was developed to assess mitochondrial aspartyl-tRNA synthetase enzyme activity in cells. Using a fluorescence reporter system we screened for drugs that modulate DARS2 splicing. Clinical information of 66 patients was obtained. The clinical severity varied from infantile onset, rapidly fatal disease to adult onset, slow and mild disease. The most common phenotype was characterized by childhood onset and slow neurological deterioration. Full wheelchair dependency was rare and usually began in adulthood. In total, 60 different DARS2 mutations were identified, 13 of which have not been reported before. Except for 4 of 42 cases published by others, all patients were compound heterozygous. Ninety-four per cent of the patients had a splice site mutation in intron 2. The groups of patients sharing the same two mutations were too small for formal assessment of genotype-phenotype correlation. However, some combinations of mutations were consistently associated with a mild phenotype. The mitochondrial aspartyl-tRNA synthetase activity was strongly reduced in patient cells. Among the compounds screened, cantharidin was identified as the most potent modulator of DARS2 splicing. In conclusion, the phenotypic spectrum of leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is wide, but most often the disease has a relatively slow and mild course. The available evidence suggests that the genotype influences the phenotype, but because of the high number of private mutations, larger numbers of patients are necessary to confirm this. The activity of mitochondrial aspartyl-tRNA synthetase is significantly reduced in patient cells. A compound screen established a 'proof of principle' that the splice site mutation can be influenced. This finding is promising for future therapeutic strategies.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Aspartato-tRNA Ligase/deficiência , Leucoencefalopatias/complicações , Leucoencefalopatias/genética , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , Adolescente , Adulto , Idade de Início , Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Cantaridina/farmacologia , Criança , Pré-Escolar , Estudos Transversais , Análise Mutacional de DNA , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Feminino , Estudos de Associação Genética , Humanos , Lactente , Leucoencefalopatias/tratamento farmacológico , Leucoencefalopatias/enzimologia , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais/tratamento farmacológico , Doenças Mitocondriais/enzimologia , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a leukodystrophy characterized by early-onset macrocephaly and delayed-onset neurological deterioration. Recessive MLC1 mutations are observed in 75% of patients with MLC. Genetic-linkage studies failed to identify another gene. We recently showed that some patients without MLC1 mutations display the classical phenotype; others improve or become normal but retain macrocephaly. To find another MLC-related gene, we used quantitative proteomic analysis of affinity-purified MLC1 as an alternative approach and found that GlialCAM, an IgG-like cell adhesion molecule that is also called HepaCAM and is encoded by HEPACAM, is a direct MLC1-binding partner. Analysis of 40 MLC patients without MLC1 mutations revealed multiple different HEPACAM mutations. Ten patients with the classical, deteriorating phenotype had two mutations, and 18 patients with the improving phenotype had one mutation. Most parents with a single mutation had macrocephaly, indicating dominant inheritance. In some families with dominant HEPACAM mutations, the clinical picture and magnetic resonance imaging normalized, indicating that HEPACAM mutations can cause benign familial macrocephaly. In other families with dominant HEPACAM mutations, patients had macrocephaly and mental retardation with or without autism. Further experiments demonstrated that GlialCAM and MLC1 both localize in axons and colocalize in junctions between astrocytes. GlialCAM is additionally located in myelin. Mutant GlialCAM disrupts the localization of MLC1-GlialCAM complexes in astrocytic junctions in a manner reflecting the mode of inheritance. In conclusion, GlialCAM is required for proper localization of MLC1. HEPACAM is the second gene found to be mutated in MLC. Dominant HEPACAM mutations can cause either macrocephaly and mental retardation with or without autism or benign familial macrocephaly.
Assuntos
Transtorno Autístico/genética , Moléculas de Adesão Celular Neuronais/genética , Deficiência Intelectual/genética , Megalencefalia/genética , Mutação , Proteínas/genética , Sequência de Aminoácidos , Animais , Transtorno Autístico/metabolismo , Encéfalo/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas de Ciclo Celular , Células Cultivadas , Cistos/genética , Cistos/metabolismo , Genes Dominantes , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Megalencefalia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas/metabolismo , Ratos , Homologia de Sequência de AminoácidosRESUMO
The autosomal recessive white matter disorder LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is caused by mutations in DARS2, coding for mtAspRS (mitochondrial aspartyl-tRNA synthetase). Generally, patients are compound heterozygous for mutations in DARS2. Many different mutations have been identified in patients, including several missense mutations. In the present study, we have examined the effects of missense mutations found in LBSL patients on the expression, enzyme activity, localization and dimerization of mtAspRS, which is important for understanding the cellular defect underlying the pathogenesis of the disease. Nine different missense mutations were analysed and were shown to have various effects on mtAspRS properties. Several mutations have a direct effect on the catalytic activity of the enzyme; others have an effect on protein expression or dimerization. Most mutations have a clear impact on at least one of the properties of mtAspRS studied, probably resulting in a small contribution of the missense variants to the mitochondrial aspartylation activity in the cell.
Assuntos
Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Mitocôndrias/enzimologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Aspartato-tRNA Ligase/deficiência , Tronco Encefálico/metabolismo , Tronco Encefálico/patologia , Células HEK293 , Humanos , Imuno-Histoquímica , Leucoencefalopatias/patologia , Mitocôndrias/metabolismo , Doenças Mitocondriais/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , TransfecçãoRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in MLC1 or GLIALCAM. The GLIALCAM gene product functions as an MLC1 beta-subunit. We aim to further clarify the molecular mechanisms of MLC caused by mutations in MLC1 or GLIALCAM. For this purpose, we analyzed a human post-mortem brain obtained from an MLC patient, who was homozygous for a missense mutation (S69L) in MLC1. We showed that this mutation affects the stability of MLC1 in vitro and reduces MLC1 protein levels in the brain to almost undetectable. However, the amount of GlialCAM and its localization were nearly unaffected, indicating that MLC1 is not necessary for GlialCAM expression or targeting. These findings were supported by experiments in primary astrocytes and in heterologous cells. In addition, we demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC.
Assuntos
Cistos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Proteínas de Membrana/genética , Mutação/genética , Proteínas/genética , Adulto , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ciclo Celular , Cistos/patologia , Evolução Fatal , Feminino , Células HEK293 , Células HeLa , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Pessoa de Meia-Idade , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Transporte Proteico , Interferência de RNA , Ratos , TransfecçãoRESUMO
LBSL (leukoencephalopathy with brain stem and spinal cord involvement and lactate elevation) is an autosomal recessive white matter disorder with slowly progressive cerebellar ataxia, spasticity and dorsal column dysfunction. Magnetic resonance imaging shows characteristic abnormalities in the cerebral white matter and specific brain stem and spinal cord tracts. LBSL is caused by mutations in the gene DARS2, which encodes mtAspRS (mitochondrial aspartyl-tRNA synthetase). The selective involvement of specific white matter tracts in LBSL is striking since this protein is ubiquitously expressed. Almost all LBSL patients have one mutation in intron 2 of DARS2, affecting the splicing of the third exon. Using a splicing reporter construct, we find cell-type-specific differences in the sensitivity to these mutations: the mutations have a larger effect on exon 3 exclusion in neural cell lines, especially neuronal cell lines, than in non-neural cell lines. Furthermore, correct inclusion of exon 3 in the normal mtAspRS mRNA occurs less efficiently in neural cells than in other cell types, and this effect is again most pronounced in neuronal cells. The combined result of these two effects may explain the selective vulnerability of specific white matter tracts in LBSL patients.
Assuntos
Processamento Alternativo/fisiologia , Aspartato-tRNA Ligase/genética , Tronco Encefálico/patologia , Ácido Láctico/metabolismo , Leucoencefalopatias/genética , Leucoencefalopatias/metabolismo , Medula Espinal/patologia , Processamento Alternativo/genética , Aspartato-tRNA Ligase/metabolismo , Tronco Encefálico/metabolismo , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Leucoencefalopatias/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Medula Espinal/metabolismo , Transfecção , Regulação para CimaRESUMO
Megalencephalic leucoencephalopathy with subcortical cysts is a genetic brain disorder with onset in early childhood. Affected infants develop macrocephaly within the first year of life, after several years followed by slowly progressive, incapacitating cerebellar ataxia and spasticity. From early on, magnetic resonance imaging shows diffuse signal abnormality and swelling of the cerebral white matter, with evidence of highly increased white matter water content. In most patients, the disease is caused by mutations in the gene MLC1, which encodes a plasma membrane protein almost exclusively expressed in brain and at lower levels in leucocytes. Within the brain, MLC1 is mainly located in astrocyte-astrocyte junctions adjacent to the blood-brain and cereborspinal fluid-brain barriers. Thus far, the function of MLC1 has remained unknown. We tested the hypothesis that MLC1 mutations cause a defect in ion currents involved in water and ion homeostasis, resulting in cerebral white matter oedema. Using whole-cell patch clamp studies we demonstrated an association between MLC1 expression and anion channel activity in different cell types, most importantly astrocytes. The currents were absent in chloride-free medium and in cells with disease-causing MLC1 mutations. MLC1-dependent currents were greatly enhanced by hypotonic pretreatment causing cell swelling, while ion channel blockers, including Tamoxifen, abolished the currents. Down regulation of endogenous MLC1 expression in astrocytes by small interfering RNA greatly reduced the activity of this channel, which was rescued by overexpression of normal MLC1. The current-voltage relationship and the pharmacological profiles of the currents indicated that the channel activated by MLC1 expression is a volume-regulated anion channel. Such channels are involved in regulatory volume decrease. We showed that regulatory volume decrease was hampered in lymphoblasts from patients with megalencephalic leucoencephalopathy. A similar trend was observed in astrocytes with decreased MLC1 expression; this effect was rescued by overexpression of normal MLC1. In the present study, we show that absence or mutations of the MLC1 protein negatively impact both volume-regulated anion channel activity and regulatory volume decrease, indicating that megalencephalic leucoencephalopathy is caused by a disturbance of cell volume regulation mediated by chloride transport.
Assuntos
Astrócitos/patologia , Cloretos/metabolismo , Cistos/fisiopatologia , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Transporte de Íons/fisiologia , Proteínas de Membrana/genética , Astrócitos/metabolismo , Tamanho Celular , Cistos/metabolismo , Cistos/patologia , Células HEK293 , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Proteínas de Membrana/metabolismoRESUMO
Mutations in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase, a key enzyme for mitochondrial translation, are correlated with leukoencephalopathy. A Ser45 to Gly45 mutation is located in the predicted targeting signal of the protein. We demonstrate in the present study, by in vivo and in vitro approaches, that this pathology-related mutation impairs the import process across mitochondrial membranes.
Assuntos
Aspartato-tRNA Ligase/genética , Aspartato-tRNA Ligase/metabolismo , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Linhagem Celular , Humanos , Leucoencefalopatias/etiologia , Leucoencefalopatias/genética , Transporte ProteicoRESUMO
Autosomal recessive mutations in eukaryotic initiation factor 2B (eIF2B) cause leukoencephalopathy vanishing white matter with a wide clinical spectrum. eIF2B comprises five subunits (α-ε; genes EIF2B1, 2, 3, 4 and 5) and is the guanine nucleotide-exchange factor (GEF) for eIF2. It plays a key role in protein synthesis. Here, we have studied the functional effects of selected VWM mutations in EIF2B2-5 by coexpressing mutated and wild-type subunits in human cells. The observed functional effects are very diverse, including defects in eIF2B complex integrity; binding to the regulatory α-subunit; substrate binding; and GEF activity. Activity data for recombinant eIF2B complexes agree closely with those for patient-derived cells with the same mutations. Some mutations do not affect these parameters even though they cause severe disease. These findings are important for three reasons; they demonstrate that measuring eIF2B activity in patients' cells has limited value as a diagnostic test; they imply that severe disease can result from alterations in eIF2B function other than defects in complex integrity, substrate binding or GEF activity, and last, the diversity of functional effects of VWM mutations implies that seeking agents to manage or treat VWM should focus on downstream effectors of eIF2B, not restoring eIF2B activity.
Assuntos
Fator de Iniciação 2B em Eucariotos/deficiência , Fator de Iniciação 2B em Eucariotos/metabolismo , Leucoencefalopatias/genética , Complexos Multiproteicos/metabolismo , Bioensaio , Extratos Celulares , Fator de Iniciação 2B em Eucariotos/química , Células HEK293 , Humanos , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Homologia de Sequência de AminoácidosRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, in the majority of cases caused by mutations in the MLC1 gene. MRI from MLC patients shows diffuse cerebral white matter signal abnormality and swelling, with evidence of increased water content. Histopathology in a MLC patient shows vacuolation of myelin, which causes the cerebral white matter swelling. MLC1 protein is expressed in astrocytic processes that are part of blood- and cerebrospinal fluid-brain barriers. We aimed to create an astrocyte cell model of MLC disease. The characterization of rat astrocyte cultures revealed MLC1 localization in cell-cell contacts, which contains other proteins described typically in tight and adherent junctions. MLC1 localization in these contacts was demonstrated to depend on the actin cytoskeleton; it was not altered when disrupting the microtubule or the GFAP networks. In human tissues, MLC1 and the protein Zonula Occludens 1 (ZO-1), which is linked to the actin cytoskeleton, co-localized by EM immunostaining and were specifically co-immunoprecipitated. To create an MLC cell model, knockdown of MLC1 in primary astrocytes was performed. Reduction of MLC1 expression resulted in the appearance of intracellular vacuoles. This vacuolation was reversed by the co-expression of human MLC1. Re-examination of a human brain biopsy from an MLC patient revealed that vacuoles were also consistently present in astrocytic processes. Thus, vacuolation of astrocytes is also a hallmark of MLC disease.
Assuntos
Astrócitos/metabolismo , Cistos/genética , Cistos/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Vacúolos/genética , Adolescente , Animais , Astrócitos/patologia , Células Cultivadas , Cistos/fisiopatologia , Regulação para Baixo/genética , Líquido Extracelular/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/fisiopatologia , Humanos , Proteínas de Membrana/fisiologia , Camundongos , Ratos , Ratos Sprague-Dawley , Vacúolos/patologiaRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive disease characterized by early infantile macrocephaly and delayed motor and cognitive deterioration. Magnetic resonance imaging (MRI) shows diffusely abnormal and swollen cerebral white matter and subcortical cysts. On follow-up, atrophy ensues. Approximately 80% of MLC patients have mutations in MLC1. We report 16 MLC patients without MLC1 mutations. Eight retained the classical clinical and MRI phenotype. The other 8 showed major MRI improvement. They lacked motor decline. Five had normal intelligence; 3 displayed cognitive deficiency. In conclusion, 2 phenotypes can be distinguished among the non-MLC1 mutated MLC patients: a classical and a benign phenotype.
Assuntos
Cistos/genética , Leucoencefalopatias/genética , Leucoencefalopatias/patologia , Proteínas de Membrana/genética , Mutação/genética , Adolescente , Adulto , Encefalopatias/complicações , Encefalopatias/patologia , Criança , Cistos/complicações , Análise Mutacional de DNA , Feminino , Seguimentos , Humanos , Leucoencefalopatias/complicações , Imageamento por Ressonância Magnética/métodos , Masculino , Estudos Retrospectivos , Adulto JovemRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy, most often caused by mutations in the MLC1 gene. MLC1 is an oligomeric plasma membrane (PM) protein of unknown function expressed mainly in glial cells and neurons. Most disease-causing missense mutations dramatically reduced the total and PM MLC1 expression levels in Xenopus oocytes and mammalian cells. The impaired expression of the mutants was verified in primary cultures of rat astrocytes, as well as human monocytes, cell types that endogenously express MLC1, demonstrating the relevance of the tissue culture models. Using a combination of biochemical, pharmacological and imaging methods, we also demonstrated that increased endoplasmatic reticulum-associated degradation and endo-lysosomal-associated degradation can contribute to the cell surface expression defect of the mutants. Based on these results, we suggest that MLC1 mutations reduce protein levels in vivo. Since the expression defect of the mutants could be rescued by exposing the mutant-protein expressing cells to low temperature and glycerol, a chemical chaperone, we propose that MLC belongs to the class of conformational diseases. Therefore, we suggest the use of pharmacological strategies that improve MLC1 expression to treat MLC patients.
Assuntos
Encefalopatias/genética , Cistos do Sistema Nervoso Central/genética , Proteínas de Membrana/genética , Mutação , Dobramento de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Astrócitos/química , Astrócitos/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Encefalopatias/metabolismo , Encefalopatias/patologia , Células Cultivadas , Cistos do Sistema Nervoso Central/metabolismo , Cistos do Sistema Nervoso Central/patologia , Expressão Gênica , Células HeLa , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Estabilidade Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Megalencephalic leukoencephalopathy with subcortical cysts is a rare leukodystrophy that is characterized by macrocephaly and a slowly progressive clinical course. It is one of the most commonly reported leukoencephalopathies in Turkey. Mutations in the MLC1 gene are the main cause of the disease. We report two patients with megalencephalic leukoencephalopathy with subcortical cysts with confirmed mutations in the MLC1 gene. The mutation in the second patient was novel. We also review identified mutations in the Turkish population.
Assuntos
Encefalopatias/genética , Cistos do Sistema Nervoso Central/genética , Leucoencefalopatias/genética , Proteínas de Membrana/genética , Mutação/genética , Encefalopatias/diagnóstico , Cistos do Sistema Nervoso Central/diagnóstico , Criança , Consanguinidade , Feminino , Humanos , Leucoencefalopatias/diagnóstico , Imageamento por Ressonância Magnética , Masculino , TurquiaRESUMO
The role of human papillomavirus type 16 (HPV16) in oral potentially malignant disorders (OPMD) and oral cavity carcinoma (OC) is still under debate. We investigated HPV16 prevalence in unstimulated saliva, oral rinse samples, oral swabs and tumour biopsies collected from OPMD (n = 83) and OC (n = 106) patients. HPV16 genotype, viral load, physical status (episomal vs. integrated) and tumour p16INK4a expression were determined. Oral HPV16 prevalence was higher in OC than in OPMD, but this difference was not statistically significant (7.5% (8/106) versus 3.6% (3/83), odds ratio (OR): 2.18, 95% confidence interval (CI): 0.56, 8.48, p = 0.26). There was a significant association (p < 0.05) between oral HPV16 infection and heavy tobacco consumption. Real-time PCR results indicated that no integration events occurred in either OPMD or OC cases based on the HPV16 E2/E6 ratio. HPV16 positive OPMD and OC patients had similar HPV16 E2 and E6 viral loads. The inter-rater agreement between tumour p16INK4a expression and oral HPV16 infection was considered as fair (k = 0.361) for OC. Our data suggest that the involvement of HPV16 in oral carcinogenesis is limited.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Papillomavirus Humano 16/genética , Neoplasias Bucais/epidemiologia , Neoplasias Bucais/virologia , Infecções por Papillomavirus/epidemiologia , Idoso , Austrália/epidemiologia , Biópsia , DNA Viral , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Razão de Chances , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Saliva/virologia , Fumar , Carga ViralRESUMO
Vanishing white matter disease is a rare neurological disease. The majority of patients reported are Caucasian individuals. We describe the first Chinese patient with typical clinical and radiological features genetically confirmed to have vanishing white matter disease for a mutation in EIF2B4, followed by a brief review of the disease.
Assuntos
Análise Mutacional de DNA , Fator de Iniciação 2B em Eucariotos/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Ácido Aspártico/análogos & derivados , Ácido Aspártico/análise , Encéfalo/patologia , Ventrículos Cerebrais/patologia , Criança , Colina/análise , Consanguinidade , Feminino , Triagem de Portadores Genéticos , Aconselhamento Genético , Genótipo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/diagnóstico , Homozigoto , Hong Kong , Humanos , Ácido Láctico/análise , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Exame Neurológico , Linhagem , FenótipoRESUMO
The "ovarioleukodystrophies" comprise a group of rare leukodystrophies associated with primary or premature ovarian failure. Some of the patients have a variant of "vanishing white matter disease" with mutations in subunits of eukaryotic initiation factor 2B (EIF2B). A 32-year-old woman who developed neurological signs related to an extensive leukoencephalopathy on magnetic resonance imaging (MRI) in the context of amenorrhea since the age of 18 years was found to be homozygous for a mutation in the EIF2B5 gene: c.338G>A/p.Arg113His. She had a progressive disease with development of tetraparesia in less than 6 years. Our observation confirms that ovarian failure in the context of a leukodystrophy warrants mutational analysis of the genes encoding the subunits of EIF2B.
Assuntos
Encefalopatias/patologia , Fator de Iniciação 2B em Eucariotos/genética , Mutação , Insuficiência Ovariana Primária/patologia , Adulto , Amenorreia/etiologia , Encefalopatias/complicações , Encefalopatias/genética , Análise Mutacional de DNA , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/complicações , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Humanos , Insuficiência Ovariana Primária/complicações , Insuficiência Ovariana Primária/genéticaRESUMO
Leukoencephalopathy with vanishing white matter (VWM) is one of the most prevalent inherited white-matter disorders, especially in Caucasian populations. VWM is unusual because of its sensitivity to febrile infections and minor head trauma. The basic defect of this enigmatic brain disease resides in the regulation of initiation of protein synthesis. Recently, undue activation of the unfolded-protein response has emerged as an important factor in the pathophysiology of VWM. Here, we discuss the mechanisms that might be responsible for the selective involvement of the brain white matter in VWM. At present, VWM research is in need of an animal model to study disease mechanisms and therapeutic interventions.
Assuntos
Encefalopatias/metabolismo , Córtex Cerebral/metabolismo , Fator de Iniciação 2B em Eucariotos/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Animais , Encefalopatias/fisiopatologia , Córtex Cerebral/patologia , Fator de Iniciação 2 em Eucariotos/fisiologia , Fator de Iniciação 2B em Eucariotos/fisiologia , Humanos , Modelos Biológicos , Biossíntese de ProteínasRESUMO
The cap-binding eukaryotic initiation factor eIF4E is phosphorylated by the mitogen-activated protein (MAP) kinase-interacting kinases (Mnk's). Three forms of the Mnk's exist in human cells: Mnk1, Mnk2a, and Mnk2b. These last two are derived from the same gene by alternative splicing and differ only at their C termini. While Mnk2a contains a MAP kinase-binding site in this region, Mnk2b lacks such a sequence and is much less readily activated by MAP kinases in vitro. Expression of Mnk2b in mammalian cells leads to increased phosphorylation of eIF4E, showing that it acts as an eIF4E kinase in vivo. While Mnk2a is cytoplasmic, a substantial amount of Mnk2b is found in the nucleus. Both enzymes contain a stretch of basic residues in their N termini that plays a role in binding to eIF4G and functions as a nuclear localization signal. Binding of eIF4G or nuclear import appears to be regulated by the C terminus of Mnk2a. Furthermore, the MAP kinase-binding site of Mnk2a regulates nuclear entry. Within the nucleus, Mnk2b and certain variants of Mnk2a that are present in the nucleus colocalize with the promyelocytic leukemia protein PML, which also binds to eIF4E.