RESUMEN
Introdução: A displasia fibrosa (DF) do osso é uma desordem congênita, rara, que corresponde de 5 a 10% dos tumores ósseos benignos, não hereditária, que cursa com amplo espectro de apresentação, variando do assintomático à dor óssea, fraturas de repetição, deformidades ósseas (fêmur em cajado de pastor e fácies leonina) e compressão de nervos cranianos. Histologicamente é composta de estroma fibroso celular de baixo a moderado grau circundando trabéculas ósseas de formato irregular sem borda osteoblástica. Todos os casos contêm a mutação GNAS1. A DF apresenta duas formas: a monostótica, mais comum (70-80%), e a poliostótica, mais rara (20-30%), que quando acompanhada de manchas café-com-leite e puberdade precoce constitui a síndrome de McCune-Albright ou Síndrome de Mazabraud em casos mais raros. O tratamento pode ser feito com medicamentos como bifosfonato ou de forma cirúrgica, objetivando-se a correção das lesões com curetagem e enxertia óssea ou como iremos mostrar a seguir, pela Técnica de Masquelet. Este trabalho relata o caso de um menino de 20 anos de idade cujos sinais e sintomas conduziam ao diagnóstico de DF sendo realizado tratamento com Técnica de Masquelet e follow up de 18 meses. Além disso, faz revisão de literatura sobre uma doença pouco comum, com variada gama de diagnósticos diferenciais. Objetivo: relatar um caso de displasia fibrosa com tratamento cirúrgico de enxerto autólogo de fíbula pela Técnica de Masquelet. Método: relato de caso de paciente do Ambulatório de Especialidade do Hospital do Servidor Público Municipal, de 20 anos de idade, que foi acompanhado por 1 ano e meio apresentando um tumor ósseo na tíbia compatível com diagnóstico de displasia fibrosa, que ao longo desse período foi submetido à Técnica de Masquelet. Conclusão: É pouco descrito na literatura o tratamento de displasia fibrosa pela Técnica de Masquelet, que mostrou ter ótimo resultado funcional para o paciente estudado. Palavras-chave: Displasia Fibrosa Óssea. Displasia Fibrosa. Técnica de Masquelet. Técnica de Membrana Induzida.
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Humanos , Masculino , Adulto , Osteomielitis/terapia , Seudoartrosis/terapia , Tibia/cirugía , Trasplante Autólogo/métodos , Huesos/fisiopatología , Fracturas Óseas/congénito , Fémur/cirugía , Displasia Fibrosa Poliostótica , Peroné/cirugía , Mutación/fisiología , Neoplasias/cirugíaRESUMEN
BACKGROUND: Evidence shows significant heterogeneity in astrocyte gene expression and function. We previously demonstrated that brain-derived neurotrophic factor (BDNF) exerts protective effects on whole brain primary cultured rat astrocytes treated with 3-nitropropionic acid (3NP), a mitochondrial toxin widely used as an in vitro model of Huntington's disease (HD). Therefore, we now investigated 3NP and BDNF effects on astrocytes from two areas involved in HD: the striatum and the entire cortex, and their involvement in neuron survival. METHODS: We prepared primary cultured rat cortical or striatal astrocytes and treated them with BDNF and/or 3NP for 24 h. In these cells, we assessed expression of astrocyte markers, BDNF receptor, and glutamate transporters, and cytokine release. We prepared astrocyte-conditioned medium (ACM) from cortical and striatal astrocytes and tested its effect on a cellular model of HD. RESULTS: BDNF protected astrocytes from 3NP-induced death, increased expression of its own receptor, and activation of ERK in both cortical and striatal astrocytes. However, BDNF modulated glutamate transporter expression differently by increasing GLT1 and GLAST expression in cortical astrocytes but only GLT1 expression in striatal astrocytes. Striatal astrocytes released higher amounts of tumor necrosis factor-α than cortical astrocytes in response to 3NP but BDNF decreased this effect in both populations. 3NP decreased transforming growth factor-ß release only in cortical astrocytes, whereas BDNF treatment increased its release only in striatal astrocytes. Finally, we evaluated ACM effect on a cellular model of HD: the rat striatal neuron cell line ST14A expressing mutant human huntingtin (Q120) or in ST14A cells expressing normal human huntingtin (Q15). Neither striatal nor cortical ACM modified the viability of Q15 cells. Only ACM from striatal astrocytes treated with BDNF and ACM from 3NP + BDNF-treated striatal astrocytes protected Q120 cells, whereas ACM from cortical astrocytes did not. CONCLUSIONS: Data suggest that cortical and striatal astrocytes respond differently to mitochondrial toxin 3NP and BDNF. Moreover, striatal astrocytes secrete soluble neuroprotective factors in response to BDNF that selectively protect neurons expressing mutant huntingtin implicating that BDNF modulation of striatal astrocyte function has therapeutic potential against neurodegeneration.
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Astrocitos/metabolismo , Factor Neurotrófico Derivado del Encéfalo/toxicidad , Corteza Cerebral/metabolismo , Cuerpo Estriado/metabolismo , Proteína Huntingtina/biosíntesis , Nitrocompuestos/toxicidad , Propionatos/toxicidad , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Cuerpo Estriado/efectos de los fármacos , Femenino , Expresión Génica , Humanos , Proteína Huntingtina/genética , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación/efectos de los fármacos , Mutación/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuroprotección/efectos de los fármacos , Neuroprotección/fisiología , Ratas , Ratas WistarRESUMEN
Ca2+ flux into axon terminals via P-/Q-type CaV2.1 channels is the trigger for neurotransmitter vesicle release at neuromuscular junctions (NMJs) and many central synapses. Recently, an arginine to proline substitution (R1673P) in the S4 voltage-sensing helix of the fourth membrane-bound repeat of CaV2.1 was linked to a severe neurological disorder characterized by generalized hypotonia, ataxia, cerebellar atrophy, and global developmental delay. The R1673P mutation was proposed to cause a gain of function in CaV2.1 leading to neuronal Ca2+ toxicity based on the ability of the mutant channel to rescue the photoreceptor response in CaV2.1-deficient Drosophila cacophony larvae. Here, we show that the corresponding mutation in rat CaV2.1 (R1624P) causes a profound loss of channel function; voltage-clamp analysis of tsA-201 cells expressing this mutant channel revealed an â¼25-mV depolarizing shift in the voltage dependence of activation. This alteration in activation implies that a significant fraction of CaV2.1 channels resident in presynaptic terminals are unlikely to open in response to an action potential, thereby increasing the probability of synaptic failure at both NMJs and central synapses. Indeed, the mutant channel supported only minimal Ca2+ flux in response to an action potential-like waveform. Application of GV-58, a compound previously shown to stabilize the open state of wild-type CaV2.1 channels, partially restored Ca2+ current by shifting mutant activation to more hyperpolarizing potentials and slowing deactivation. Consequently, GV-58 also rescued a portion of Ca2+ flux during action potential-like stimuli. Thus, our data raise the possibility that therapeutic agents that increase channel open probability or prolong action potential duration may be effective in combatting this and other severe neurodevelopmental disorders caused by loss-of-function mutations in CaV2.1.
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Canales de Calcio Tipo N/genética , Activación del Canal Iónico/genética , Mutación/genética , Trastornos del Neurodesarrollo/genética , Potenciales de Acción/genética , Potenciales de Acción/fisiología , Animales , Calcio/metabolismo , Activación del Canal Iónico/fisiología , Mutación/fisiología , Trastornos del Neurodesarrollo/fisiopatología , Unión Neuromuscular/genética , Unión Neuromuscular/fisiopatología , Neuronas/fisiología , Técnicas de Placa-Clamp/métodos , Terminales Presinápticos/fisiología , Conejos , Ratas , Sinapsis/genética , Transmisión Sináptica/genética , Transmisión Sináptica/fisiologíaRESUMEN
Introduction: In mammals, there is evidence that glutamate has a role as a wake-active neurotransmitter. So using video-based analysis of Drosophila behavior, we undertook a study to examine if glutamate, which has been previously shown to have an excitatory role in neuromuscular junctions in Drosophila, may have a conserved wake-active role in the adult brain. Aims and Methods: Using 6- to 9-day-old female flies, we examined the effect of perturbations of the glutamatergic signaling on total wakefulness and wake bout architecture. We increased and decreased neuronal activity of glutamatergic neurons in the brains of adult flies using Upstream Activating Sequence (UAS) NaChBac and UAS EKO, respectively. We blocked neurotransmission from glutamatergic neurons in adult flies using the UAS-driven temperature-sensitive dynamin mutation shibirets. We examined the behavior of flies with loss of function mutations of individual subunits of brain-specific ionotropic glutamate receptors. Results: Increasing the activity of glutamatergic neurons in the adult brain led to a significant increase in wakefulness compared to the control groups both in the daytime and nighttime and decreasing the activity of these same neurons reduced wakefulness in the nighttime. Blocking neurotransmitter release in glutamatergic neurons significantly reduced wake in the nighttime. The ionotropic receptor mutants had significantly less wake in the nighttime than their respective genetic background controls. Conclusion: The results show the following: glutamate is indeed a wake-active neurotransmitter in Drosophila; there is a major time of day effect associated with loss of glutamatergic neurotransmission; and it is a major wake-active neurotransmitter in the nighttime.
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Drosophila melanogaster/fisiología , Ácido Glutámico/fisiología , Neurotransmisores/fisiología , Sueño/fisiología , Vigilia/fisiología , Animales , Animales Modificados Genéticamente , Encéfalo/fisiología , Femenino , Locomoción/fisiología , Mutación/fisiología , Neuronas/fisiología , Transducción de Señal/fisiología , Grabación en VideoRESUMEN
INTRODUÇÃO: O HTLV-1 é o agente etiológico de doenças humanas, tais como leucemia/linfoma de célula T do adulto (ATLL), paraparesia espástica tropical/mielopatia associada ao HTLV (HAM/TSP), dermatite infectiva associada ao HTLV-1 (DIH), entre outras. Estima-se que cerca de 5-10 milhões de pessoas estejam infectadas pelo HTLV-1 no mundo e apesar dessa infecção ser endêmica em diferentes regiões geográficas, ainda permanece sem métodos terapêuticos eficazes. O genoma desse retrovírus é composto por duas fitas simples de RNA, com os genes gag, pol, env e uma região próxima à extremidade 3' conhecida como pX. A região pX contém quatro quadros abertos de leitura (ORFs) que codificam proteínas regulatórias específicas. A ORF-I codifica as proteínas p12 e p8, que quando expressas em quantidades equivalentes favorecem a infectividade e persistência viral. Mutações gênicas específicas na ORF-I do HTLV-1 podem alterar o padrão de expressão e, consequentemente, a concentração relativa destas proteínas, implicando na alteração da persistência viral e no desfecho da infecção. OBJETIVO: Caracterizar a ORF-I do HTLV-1 de pacientes com diferentes perfis clínicos. MATERIAL E MÉTODOS: Inicialmente foi realizada a anotação completa do principal genoma do HTLV-1 (ATK1), utilizado como sequência referência para a caracterização molecular da ORF-I. Em seguida, 1530 sequências da ORF-I provenientes de indivíduos assintomáticos e com HAM/TSP foram submetidas a análise de dataming para busca de associações entre mutações, carga proviral e sintomatologia. Para avaliar o grau de conservação genética da ORF-I em outros perfis clínicos, amostras de 23 pacientes assintomáticos, 11 pacientes com DIH, 13 com ATLL e 16...
INTRODUTION: The HTLV-1 is the etiological agent of some human diseases, such asadult T-cell leukaemia/lymphoma (ATLL), HTLV-1-associated myelopathy/tropicalspastic paraparesis (HAM/TSP), infective dermatitis associated to HTLV-1 (IDH),among others. It is estimated that approximately 5-10 million people are infected withHTLV-1 in the world and although this infection is endemic in different geographicregions, it still remains without effective therapeutic methods. The genome of thisretrovirus is composed of two single strands of RNA, with the genes gag, pol, env and aregion near the 3' end, known as pX. The pX region contains four open reading frames(ORFs) that encode specific regulatory proteins. The ORF-I encodes the p12 and p8proteins, which when expressed in equivalent concentrations favor infectivity and viralpersistence. Specific mutations in HTLV-1 ORF-I may alter the expression and,consequently, the relative concentration of these proteins, implying in viral persistence alteration and outcome of infection. OBJECTIVE: Characterize the HTLV-1 ORF-Ifrom patients with different clinical profiles. MATERIAL AND METHODS: First, thecomplete annotation of the main genome of HTLV-1 (ATK1), used as a referencesequence for the molecular characterization of ORF-I, was initially performed. Then,1530 ORF-I sequences from asymptomatic and HAM/TSP individuals were submitted todataming analysis to search associations. To assess the ORF-I genetic conservation statusin other clinical profiles, samples from 23 asymptomatic patients, 11 patients with IDH,13 with ATLL and 16 with...
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Humanos , Mutación/fisiología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Virus Linfotrópico T Tipo 1 Humano/patogenicidadRESUMEN
The Li-Fraumeni syndrome is characterized clinically by the appearance of tumors in multiple organs generally at an early age. This hereditary condition is caused by germinal mutations in the TP53 gene, which codifies for the tumoural suppressor gene p53. We present the case of a patient aged 31 with clinical and molecular diagnosis of Li-Fraumeni syndrome who presented two synchronous tumors: a leiomyosarcoma on the forearm and a phyllodes breast tumour. She had a family history of cancer, including a son diagnosed with a cortical adrenal carcinoma when he was three years old, who died at five from the disease. Furthermore, her maternal grandmother and great-grandmother died of stomach cancer at 56 and 60 years old, respectively, while her other great-grandmother and a great aunt presented with breast cancer at the ages of 60 and 40, respectively. After genetic counseling, complete sequencing and analysis of duplications and deletions in the TP53 gene were ordered prior to diagnosis. The molecular analysis of a DNA sample taken from peripheral blood lymphocytes revealed the germinal mutation c.527G>T (p.Cys176Phe) on exon 5 of the TP53 gene, a deleterious mutation described previously in tumoural tissues. To our knowledge, this is the first published case in Colombia of Li-Fraumeni syndrome with confirmed molecular diagnosis. The diagnosis and management of Li-Fraumeni syndrome should be performed by a multidisciplinary team, and genetic counselling should be offered to patients and their relatives.
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Neoplasias de la Mama/diagnóstico , Exones/genética , Exones/fisiología , Genes p53/genética , Genes p53/fisiología , Síndrome de Li-Fraumeni , Mutación/genética , Mutación/fisiología , Neoplasias Gástricas/diagnóstico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Humanos , Proteína p53 Supresora de Tumor/químicaRESUMEN
The recent finding that the human version of a neurodevelopmental enhancer of the Wnt receptor Frizzled 8 (FZD8) gene alters neural progenitor cell cycle timing and brain size is a step forward to understanding human brain evolution. The human brain is distinctive in terms of its cognitive abilities as well as its susceptibility to neurological disease. Identifying which of the millions of genomic changes that occurred during human evolution led to these and other uniquely human traits is extremely challenging. Recent studies have demonstrated that many of the fastest evolving regions of the human genome function as gene regulatory enhancers during embryonic development and that the human-specific mutations in them might alter expression patterns. However, elucidating molecular and cellular effects of sequence or expression pattern changes is a major obstacle to discovering the genetic bases of the evolution of our species. There is much work to do before human-specific genetic and genomic changes are linked to complex human traits.
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Evolución Biológica , Encéfalo/metabolismo , Mutación/fisiología , Animales , Secuencia de Bases , Elementos de Facilitación Genéticos , Predisposición Genética a la Enfermedad , Especiación Genética , Genoma Humano , Humanos , Enfermedades del Sistema Nervioso/genética , Carácter Cuantitativo HeredableRESUMEN
OBJECTIVE: To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. METHODS: Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson's trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. RESULTS: Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). CONCLUSION: This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis.
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Modelos Animales de Enfermedad , Endopeptidasas/genética , Endopeptidasas/metabolismo , Riñón/metabolismo , Riñón/patología , Mutación/fisiología , Superóxidos/metabolismo , Animales , Apoptosis/genética , Apoptosis/fisiología , Colágeno/análisis , Creatinina/sangre , Eritropoyetina/análisis , Femenino , Fibrosis/genética , Fibrosis/metabolismo , Metaloproteinasa 9 de la Matriz/análisis , Ratones , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo , Superóxido Dismutasa/análisis , Superóxido Dismutasa-1 , Superóxidos/análisis , Factor de Crecimiento Transformador beta/análisisRESUMEN
Objective To establish whether the mutation in the Immp2L gene induces renal fibrosis and whether aging exacerbates renal morphology in mice. Methods Female mutant mice with mutation in the inner mitochondrial membrane peptidase 2-like protein at 3 and 18 months of age were used. Renal fibrosis was analyzed using classic fibrosis score, Masson’s trichrome staining, and analysis of profibrotic markers using real time polymerase chain reaction (superoxide dismutase 1, metalloproteinase-9, erythropoietin, transforming growth factor beta), and immunostaining (fibroblasts and Type IV collagen). Oxidative stress markers were determined by immunohistochemistry. The number of renal apoptotic cells was determined. Renal function was estimated by serum creatinine. Results Young mutant mice had significantly more glomerulosclerosis than age-matched mice (p=0.034). Mutant mice had more tubular casts (p=0.025), collagen deposition (p=0.019), and collagen type IV expression (p<0.001). Superoxide dismutase 1 expression was significantly higher in young mutants (p=0.038). Old mutants exhibited significantly higher expression of the fibroblast marker and macrophage marker (p=0.007 and p=0.012, respectively). The real time polymerase chain reaction of metalloproteinase-9 and erythropoietin were enhanced 2.5- and 6-fold, respectively, in old mutants. Serum creatinine was significantly higher in old mutants (p<0.001). Conclusion This mutation altered renal architecture by increasing the deposition of extracellular matrix, oxidative stress, and inflammation, suggesting a protective role of Immp2L against renal fibrosis. .
Objetivo Estabelecer se a mutação no gene Immp2L induz à fibrose renal e se o envelhecimento exacerba a morfologia renal em camundongos. Métodos Foram usadas fêmeas de camundongos mutantes para proteína semelhante à peptidase 2 da camada interna da mitocôndria, com 3 e 18 meses de idade. Para analisar a fibrose renal, foram usados o escore clássico de fibrose, a coloração com tricrômio de Masson, e a análise de marcadores profibróticos, por meio da reação em cadeia de polimerase em tempo real (superóxido dismutase 1, metalonoproteinase-9, eritropoietina e fator transformador de crescimento beta), e a imunocoloração (fibroblastos e colágeno IV). Marcadores de estresse oxidativo foram determinados por imuno-histoquímica. O número de células apoptóticas renais foi analisado. A função renal foi estimada por creatinina sérica. Resultados Camundongos mutantes jovens apresentaram glomeruloesclerose em quantidade significativamente maior que animais da mesma idade (p=0,034). Os mutantes mostraram maior formação de cilindros tubulares (p=0,025), deposição de colágeno (p=0,019) e maior expressão de colágeno do tipo IV (p<0,001). A expressão de superóxido dismutase 1 foi maior em mutantes jovens (p=0,038). Mutantes idosas exibiram maior expressão dos marcadores de fibroblastos e macrófagos (p=0,007 e p=0,012, respectivamente). As reações da cadeia de polimerase em tempo real da metalanoproteinase-9 e da eritropoietina estavam aumentadas em 2,5 e 6 vezes, respectivamente, em mutantes idosas. A creatinina sérica foi significantemente maior em animais idosos mutantes (p<0,001). Conclusão Essa mutação alterou a arquitetura renal pelo aumento da deposição de matriz extracelular, estresse oxidativo e inflamação, sugerindo papel de proteção de Immp2L contra a fibrose renal. .
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Animales , Femenino , Ratones , Modelos Animales de Enfermedad , Endopeptidasas/genética , Endopeptidasas/metabolismo , Riñón/metabolismo , Riñón/patología , Mutación/fisiología , Superóxidos/metabolismo , Apoptosis/genética , Apoptosis/fisiología , Colágeno/análisis , Creatinina/sangre , Eritropoyetina/análisis , Fibrosis/genética , Fibrosis/metabolismo , Metaloproteinasa 9 de la Matriz/análisis , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Insuficiencia Renal Crónica/genética , Insuficiencia Renal Crónica/metabolismo , Superóxido Dismutasa/análisis , Superóxidos/análisis , Factor de Crecimiento Transformador beta/análisisRESUMEN
Thyroid hormone action is predominantly mediated by thyroid hormone receptors (THRs), which are encoded by the thyroid hormone receptor α (THRA) and thyroid hormone receptor ß (THRB) genes. Patients with mutations in THRB present with resistance to thyroid hormone ß (RTHß), which is a disorder characterized by elevated levels of thyroid hormone, normal or elevated levels of TSH and goitre. Mechanistic insights about the contributions of THRß to various processes, including colour vision, development of the cochlea and the cerebellum, and normal functioning of the adult liver and heart, have been obtained by either introducing human THRB mutations into mice or by deletion of the mouse Thrb gene. The introduction of the same mutations that mimic human THRß alterations into the mouse Thra and Thrb genes resulted in distinct phenotypes, which suggests that THRA and THRB might have non-overlapping functions in human physiology. These studies also suggested that THRA mutations might not be lethal. Seven patients with mutations in THRα have since been described. These patients have RTHα and presented with major abnormalities in growth and gastrointestinal function. The hypothalamic-pituitary-thyroid axis in these individuals is minimally affected, which suggests that the central T3 feedback loop is not impaired in patients with RTHα, in stark contrast to patients with RTHß.
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Receptores de Hormona Tiroidea/genética , Receptores de Hormona Tiroidea/metabolismo , Enfermedades de la Tiroides/genética , Enfermedades de la Tiroides/metabolismo , Hormonas Tiroideas/genética , Hormonas Tiroideas/metabolismo , Animales , Humanos , Mutación/fisiología , Enfermedades de la Tiroides/diagnósticoRESUMEN
Los nevos epidérmicos (NE) son hamartomas cutáneos de baja frecuencia originados en células pluripotenciales del ectodermo embrionario. Se reconocen diferentes variantes según su morfología y topografía. En 2012 se introdujo una nueva forma clínica con característicassingulares: el RAVEN, acrónimo de rounded and velvety epidermal nevus.Presentamos el primer caso argentino de esta variedad de nevo epidérmico y resaltamos sus características principales...
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Humanos , Acantosis Nigricans , Hamartoma , Mutación/fisiología , Nevo/diagnósticoRESUMEN
OBJECTIVE: To investigate the role of KAL1 abnormalities in Brazilian patients with Kallmann syndrome. DESIGN: In vitro experiments. SETTING: Academic medical center. PATIENT(S): One hundred fifteen Brazilian patients (98 men) with Kallmann syndrome. INTERVENTION(S): Peripheral blood leukocytes were used to obtain DNA. MAIN OUTCOME MEASURE(S): Direct sequencing and multiplex ligation-dependent probe amplification were used to identify KAL1 abnormalities. RESULT(S): We identified four KAL1 mutations (p.Met1?, p.Ala33Glyfs, p.Arg257*, and p.Trp462*) and two multiple exon deletions (exons 1-2 and 3-14) in six new male patients. Overall, 17 KAL1 defects (14.8%) were identified in the entire cohort of patients with Kallmann syndrome, including previously studied cases. KAL1-mutated patients presented with a more severe reproductive and nonreproductive phenotype (synkinesia, renal malformations, cryptorchidism, and anatomic olfactory abnormalities) in comparison with patients without KAL1 mutations. Intragenic deletions were one of the most often encountered defects (29.4%). These deletions can be missed by polymerase chain reaction (PCR) due to Yq11.2 KAL1 pseudogene (KALP) spurious amplification. CONCLUSION(S): These results indicate that intragenic multiexon deletions are one of the most frequent KAL1 abnormalities, which can be more accurately detected by multiplex ligation-dependent probe amplification. In addition, KAL1 sequencing results should be interpreted with caution, and stringency conditions of the PCR reaction should be adjusted to avoid pseudogene amplification.
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Análisis Mutacional de ADN/métodos , Proteínas de la Matriz Extracelular/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Síndrome de Kallmann/genética , Reacción en Cadena de la Polimerasa Multiplex , Proteínas del Tejido Nervioso/genética , Adulto , Automatización , Secuencia de Bases , Análisis Mutacional de ADN/instrumentación , Femenino , Frecuencia de los Genes , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Humanos , Síndrome de Kallmann/diagnóstico , Síndrome de Kallmann/epidemiología , Masculino , Mutación/genética , Mutación/fisiología , Prevalencia , Seudogenes/genéticaRESUMEN
In plants, mitochondrial sequence tandem repeats (STRs) have been associated with intragenomic recombination, a process held responsible for evolutionary outcomes such as gene regulation or cytoplasmic male-sterility. However, no link has been established between the recurrent accumulation of STRs and increased mutation rates in specific regions of the plant mtDNA genome. Herein, we surveyed this possibility by comparing, in a phylogenetic context, the variation of a STR-rich mitochondrial intron (nad5-4) with eleven mtDNA genes devoid of STRs within Abies (Pinaceae) and its related genera. This intron has been accumulating repeated stretches, generated by at least three-independent insertions, before the split of the two Pinaceae subfamilies, Abietoideae and Pinoideae. The last of these insertions occurred before the divergence of Abies and produced, exclusively within this genus, a tenfold increase of both the indel and substitution rates in the STR hotspot of the intron. The regions flanking the STRs harbored mutation rates as low as those estimated in mitochondrial genes devoid of repeated stretches. Further searches in complete plant mtDNA genomes, and previous studies reporting polymorphic mtSTRs, revealed that repeated stretches are common in all sorts of plants, but their accumulation in STR hotspots appears to be taxa specific. Our study suggests a new mutagenic role for repeated sequences in the plant mtDNA.
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Abies/genética , Evolución Molecular , Genoma Mitocondrial/genética , Genoma de Planta , Repeticiones de Microsatélite/genética , Secuencia de Bases , Genes Mitocondriales , Genoma de Planta/genética , Mutagénesis Insercional/genética , Mutagénesis Insercional/fisiología , Mutación/fisiología , Filogenia , Pinaceae/clasificación , Pinaceae/genéticaRESUMEN
Presenilin 1 (PS1) mutations are the most common cause of early-onset familial Alzheimer's disease (EOFAD). They show a common phenotypic profile characterized by early age of onset, severe dementia and distinct neurodegeneration. The largest population of EOFAD carries the E280A mutation in PS1 and resides in Antioquia, Colombia, currently comprising around 5,000 individuals. Carriers start showing memory impairment in the third decade of life, followed by progressive impairment of language and other cognitive processes. They reach mild cognitive impairment around 45 and dementia around 50 years of age. There is some phenotypic variability among the carriers of this single PS1 mutation. Some patients present with epilepsy, verbal impairment, and cerebellar ataxia. Neuropathologically, PS1 E280A cases show pronounced brain atrophy, severe amyloid-ß pathology, distinct hyperphosphorylated tau-related pathology, and cerebellar damage. The earliest event identified by functional magnetic imaging resonance is hyperactivation within the right anterior hippocampus around 33 years of age. This well-studied population with a clear pre-clinical profile and wide phenotypic variability in age of onset and clinical presentation is ideally suited for clinical trials and to study molecular mechanisms of Alzheimer's disease.
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Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/psicología , Mutación/genética , Presenilina-1/genética , Adulto , Edad de Inicio , Anciano , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Encéfalo/patología , Cognición/fisiología , Colombia/epidemiología , Heterocigoto , Humanos , Persona de Mediana Edad , Mutación/fisiología , Fenotipo , Presenilina-2/genéticaRESUMEN
The tumor suppressor TP53 gene is one of the most frequently mutated in different types of human cancer. Particularly in colorectal cancer (CRC), it is believed that TP53 mutations play a role in the adenoma-carcinoma transition of tumors during pathological process. In order to analyze TP53 expressed alleles in CRC, we examined TP53 mRNA in tumor samples from 101 patients with sporadic CRC. Samples were divided in two groups defined according to whether they exhibit positive or negative P53 protein expression as detected by immunohistochemistry (IHC). The presence of TP53 mutation was a common event in tumors with an overall frequency of 54.5%. By direct sequencing, we report 42 different TP53 sequence changes in 55 CRC patients, being two of them validated polymorphisms. TP53 mutations were more frequent in positive than in negative P53 detection group (p<0.0001), being the precise figures 79.6% and 30.8%, respectively. In addition, the mutation profiles were also different between the two groups of samples; while most of the mutations detected in P53 positive group were missense (38 out of 39), changes in P53 negative detection group include 7 insertions/deletions, 6 missense, 2 nonsense and 1 silent mutation. As previously observed, most mutations were concentrated in regions encoding P53 DNA binding domain (DBD). Codons 175, 248 and 273 together account for 36.7% of point mutations, in agreement with previous observations provided that these codons are considered mutation hotspots. Interestingly, we detected two new deletions and two new insertions. In addition, in three samples we detected two deletions and one insertion that could be explained as putative splicing variants or splicing errors.
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Carcinoma/genética , Neoplasias Colorrectales/genética , Mutación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Adenoma/genética , Adenoma/metabolismo , Secuencia de Bases , Carcinoma/metabolismo , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Masculino , Análisis por Apareamiento , Datos de Secuencia Molecular , Mutación/fisiología , Dominios y Motivos de Interacción de Proteínas/genética , Procesamiento Proteico-Postraduccional/genética , Proteolisis , Proteína p53 Supresora de Tumor/químicaRESUMEN
The present study was performed to establish the prevalence of the recurrent fusion transcripts in Argentinean pediatric patients with acute lymphoblastic leukemia (ALL). A total of 380 newly diagnosed children (including 50 infants and 44 T-ALL) were screened by RT-PCR; the incidence of recurrent rearrangements was: ETV6-RUNX1, 12.9%; TCF3-PBX1, 5.0%; BCR-ABL1, 1.6%; and MLL rearrangements, 10.5%. STIL-TAL1 was detected in 22.7% of T-ALL cases. In B-ALL cases, the pEFS was significantly influenced by the presence of genetic alterations. RT-PCR studies improved patients' stratification and also the overall outcome of children treated in a pediatric hospital from a developing country.
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Técnicas de Diagnóstico Molecular/métodos , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adolescente , Argentina/epidemiología , Niño , Preescolar , Análisis Citogenético , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Técnicas de Diagnóstico Molecular/estadística & datos numéricos , Mutación/fisiología , Tasa de Mutación , Pediatría/métodos , Pediatría/estadística & datos numéricos , Leucemia-Linfoma Linfoblástico de Células Precursoras/epidemiología , Recurrencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricosRESUMEN
Metallo-ß-lactamases (MßLs) represent one of the main mechanisms of bacterial resistance against ß-lactam antibiotics. The elucidation of their mechanism has been limited mostly by the structural diversity among their active sites. All MßLs structurally characterized so far present a Cys or a Ser residue at position 221, which is critical for catalysis. GOB lactamases stand as an exception within this picture, possessing a Met residue in this location. We studied different mutants in this position, and we show that Met221 is essential for protein stability, most likely due to its involvement in a hydrophobic core. In contrast to other known MßLs, residue 221 is not involved in metal binding or in catalysis in GOB enzymes, further highlighting the structural diversity of MßLs. We also demonstrate the usefulness of protein periplasmic profiles to assess the contribution of protein stability to antibiotic resistance.
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Metionina/genética , beta-Lactamasas/genética , Sustitución de Aminoácidos , Antibacterianos/farmacología , Farmacorresistencia Bacteriana/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/patogenicidad , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Mutación/genética , Mutación/fisiología , Plásmidos/genética , Conformación Proteica , beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
During tissue morphogenesis and differentiation, cells must self-renew while contemporaneously generating daughters that contribute to the growing tissue. How tissues achieve this precise balance between proliferation and differentiation is, in most instances, poorly understood. This is in part due to the difficulties in dissociating the mechanisms that underlie tissue patterning from those that regulate proliferation. In the migrating posterior lateral line primordium (PLLP), proliferation is predominantly localised to the leading zone. As cells emerge from this zone, they periodically organise into rosettes that subsequently dissociate from the primordium and differentiate as neuromasts. Despite this reiterative loss of cells, the primordium maintains its size through regenerative cell proliferation until it reaches the tail. In this study, we identify a null mutation in the Wnt-pathway transcription factor Lef1 and show that its activity is required to maintain proliferation in the progenitor pool of cells that sustains the PLLP as it undergoes migration, morphogenesis and differentiation. In absence of Lef1, the leading zone becomes depleted of cells during its migration leading to the collapse of the primordium into a couple of terminal neuromasts. We show that this behaviour resembles the process by which the PLLP normally ends its migration, suggesting that suppression of Wnt signalling is required for termination of neuromast production in the tail. Our data support a model in which Lef1 sustains proliferation of leading zone progenitors, maintaining the primordium size and defining neuromast deposition rate.
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Proliferación Celular , Homeostasis/genética , Sistema de la Línea Lateral/embriología , Factores de Transcripción/fisiología , Proteínas Wnt/fisiología , Proteínas de Pez Cebra/fisiología , beta Catenina/fisiología , Aletas de Animales/embriología , Aletas de Animales/crecimiento & desarrollo , Aletas de Animales/metabolismo , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Embrión no Mamífero , Homeostasis/fisiología , Sistema de la Línea Lateral/metabolismo , Masculino , Morfogénesis/genética , Morfogénesis/fisiología , Mutación/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Pez Cebra/embriología , Pez Cebra/genética , Pez Cebra/metabolismo , Pez Cebra/fisiología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
CONTEXT: CTNNB1/ß-catenin mutations and activation of Wnt/ß-catenin pathway are frequent in adult adrenocortical tumors (ACT), but data on childhood ACT are lacking. OBJECTIVE: The aim of the study was to investigate the presence of Wnt/ß-catenin pathway abnormalities in childhood ACT. PATIENTS AND METHODS: Clinicopathological findings and outcome of 62 childhood ACT patients were analyzed regarding CTNNB1 mutations and the expression of Wnt-related genes (CTNNB1; WNT4, a Wnt ligand; SFRP1, DKK3, and AXIN1, Wnt inhibitors; TCF7, a transcription factor; and MYC and WISP2, target genes) by quantitative PCR and immunohistochemistry. RESULTS: CTNNB1-activating mutations were found in only four of 62 ACT (6%), all of them harboring TP53 mutation. There was association between the presence of CTNNB1 mutations and death (P = 0.02). Diffuse ß-catenin accumulation was found in 71% of ACT, even in ACT without CTNNB1 mutations. Compared to normal adrenals, ACT presented increased expression of CTNNB1 (P = 0.008) and underexpression of Wnt inhibitor genes: DKK3 (P < 0.0001), SFRP1 (P = 0.05), and AXIN1 (P = 0.04). With regard to Wnt/ß-catenin target genes, ACT presented increased expression of WISP2 but lower expression of MYC. Higher overall survival was associated with underexpression of SFRP1 (P = 0.01), WNT4 (P = 0.004), and TCF7 (P < 0.01). CONCLUSIONS: CTNNB1 mutations are not common in childhood ACT but appear to associate with poor prognosis. Nevertheless, most ACT exhibit increased expression of ß-catenin and WISP2 and reduced expression of Wnt inhibitor genes (DKK3, SFRP1, and AXIN1). Thus, in addition to CTNNB1 mutations, other genetic events affecting the Wnt/ß-catenin pathway may be involved in childhood adrenocortical tumorigenesis.
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Neoplasias de la Corteza Suprarrenal/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/fisiología , beta Catenina/fisiología , Adolescente , Neoplasias de la Corteza Suprarrenal/genética , Proteína Axina/fisiología , Proteínas CCN de Señalización Intercelular , Niño , Preescolar , Estudios de Cohortes , ADN/genética , ADN/aislamiento & purificación , Femenino , Humanos , Inmunohistoquímica , Lactante , Péptidos y Proteínas de Señalización Intercelular/fisiología , Masculino , Mutación/fisiología , Proteínas Proto-Oncogénicas c-myc/fisiología , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Proteínas Represoras , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Análisis de Supervivencia , Factor 1 de Transcripción de Linfocitos T/fisiología , Factores de Transcripción/fisiología , Resultado del Tratamiento , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Wnt/antagonistas & inhibidores , Proteínas Wnt/genética , Proteína Wnt4/fisiología , beta Catenina/genéticaRESUMEN
BACKGROUND: The intravenous anesthetic propofol acts as a positive allosteric modulator of glycine (GlyRs) and γ-aminobutyric acid type A (GABAARs) receptors. Although the role of transmembrane residues is recognized, little is known about the involvement of other regions in the modulatory effects of propofol. Therefore, the influence of the large intracellular loop in propofol sensitivity of both receptors was explored. METHODS: The large intracellular loop of α1 GlyRs and α1ß2 GABAARs was screened using alanine replacement. Sensitivity to propofol was studied using patch-clamp recording in HEK293 cells transiently transfected with wild type or mutant receptors. RESULTS: Alanine mutation of a conserved phenylalanine residue within the α1 large intracellular loop significantly reduced propofol enhancement in both GlyRs (360 ± 30 vs. 75 ± 10%, mean ± SEM) and GABAARs (361 ± 49% vs. 80 ± 23%). Remarkably, propofol-hyposensitive mutant receptors retained their sensitivity to other allosteric modulators such as alcohols, etomidate, trichloroethanol, and isoflurane. At the single-channel level, the ability of propofol to increase open probability was significantly reduced in both α1 GlyR (189 ± 36 vs. 22 ± 13%) and α1ß2 GABAAR (279 ± 29 vs. 29 ± 11%) mutant receptors. CONCLUSION: In this study, it is demonstrated that the large intracellular loop of both GlyR and GABAAR has a conserved single phenylalanine residue (F380 and F385, respectively) that influences its sensitivity to propofol. Results suggest a new role of the large intracellular loop in the allosteric modulation of two members of the Cys-loop superfamily. Thus, these data provide new insights into the molecular framework behind the modulation of inhibitory ion channels by propofol.