RESUMO
SUMMARY: In order to help molecular geneticists to rapidly identify CNVs responsible for inherited diseases among amplicons sequencing data generated by NGS, we designed a user-friendly tool ' Cov'Cop '. Using the run's coverage file provided by the sequencer, Cov'Cop simultaneously analyzes all the patients of the run using a two-stage algorithm containing correction and normalization levels and provides an easily understandable output, showing with various colors, potentially deleted and duplicated amplicons. AVAILABILITY AND IMPLEMENTATION: https://git.unilim.fr/merilp02/CovCop. CONTACT: asliabaldini@unilim.fr. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Variações do Número de Cópias de DNA , Análise Mutacional de DNA/métodos , Software , Algoritmos , Doenças Genéticas Inatas/genética , HumanosRESUMO
The autosomal recessive demyelinating form of Charcot-Marie-Tooth can be due to SH3TC2 gene pathogenic variants (CMT4C, AR-CMTde-SH3TC2). We report on a series of 13 patients with AR-CMTde-SH3TC2 among a French cohort of 350 patients suffering from all type of inheritance peripheral neuropathy. The SH3TC2 gene appeared to be the most frequently mutated gene for demyelinating neuropathy in this series by NGS. Four new pathogenic variants have been identified: two nonsense variants (p.(Tyr970*), p.(Trp1199*)) and two missense variants (p.(Leu1126Pro), p.(Ala1206Asp)). The recurrent variant p.Arg954* was present in 62%, and seems to be a founder mutation. The phenotype is fairly homogeneous, as all these patients, except the youngest ones, presented scoliosis and/or hearing loss.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Surdez/genética , Variação Genética/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Escoliose/genética , Adulto , Idoso , Doença de Charcot-Marie-Tooth/epidemiologia , Criança , Estudos de Coortes , Surdez/epidemiologia , Feminino , França/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Escoliose/epidemiologia , Adulto JovemRESUMO
A typical monoclonal IgG dysglobulinemia whether benign (monoclonal gammopathy of undetermined significance, MGUS) or malignant can give rise to peripheral neuropathy by damaging nerves. At first, neurotoxicity of the chemotherapy if the patient is treated must be ruled out in such cases. Indeed, a variety of other mechanisms have been described: endoneurial deposits of immunoglobulin, infiltration of the immunoglobulin within myelin sheaths, POEMS syndrome, deposits of amyloid, chronic inflammatory demyelinating polyradiculoneuropathy and infiltration of malignant cells. Ultrastructural examination of a nerve biopsy can be decisive in combination with routine histological and immunopathological examinations. Characterization of the mechanism of the neuropathy in a dysglobulinemic context is important as it governs therapeutic options, which in certain cases are particularly beneficial.
Assuntos
Imunoglobulina G/sangue , Microscopia Eletrônica/métodos , Paraproteinemias/patologia , Doenças do Sistema Nervoso Periférico/patologia , Humanos , Microscopia Eletrônica/instrumentação , Paraproteinemias/imunologia , Paraproteinemias/fisiopatologia , PlasmocitomaRESUMO
The gene therapy strategy using the hsvl-thymidine kinase gene (TK) and ganciclovir (GCV) injections that has been used for treating human glioblastomas has not been as effective as expected after the first animal experiments. A better understanding of the different steps involved in this treatment, like gene transfer, gene expression, and sensitivity of the recipient cells, is needed. After proposing sensitivity criteria for the TK/GCV system and for the bystander effect, based on the levels of GCV that can be reached in vivo, we studied seven human glioblastoma cell lines (U87, U118, U251, SNB19, SNB75, SF295, SF539) for their sensitivity to the TK/GCV system. We also studied their in vitro bystander effect and their in vitro transfectability using LipofectAMINE as a transfection enhancer. Among six human glioblastoma cell lines stably transfected with the TK gene, five were sensitive to TK/GCV, and two had a good in vitro bystander effect. The in vitro transfectability of the cell lines tested was low (< or = 1%) compared to that of an established animal cell line, C6 rat glioma, in which 20-30% of the cells can be transfected routinely. According to this in vitro analysis, most of the glioblastoma cell lines should be sensitive to the TK/GCV system, but there is an urgent need for agents to increase transfection efficiency.
Assuntos
Ganciclovir/farmacologia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Glioblastoma/terapia , Timidina Quinase/genética , Transfecção , Animais , Resinas de Troca de Cátion , Sobrevivência Celular , Genes Reporter , Vetores Genéticos , Glioblastoma/enzimologia , Glioblastoma/patologia , Histocitoquímica , Humanos , Lipídeos , Ratos , Simplexvirus/genética , Células Tumorais Cultivadas , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Previously, we described a nonviral cytoplasmic gene therapy vector system based on the T7 autogene concept. This system has been shown to achieve rapid and high levels of gene expression in a variety of animal cells and tissues. To test the utility of the system in vivo tumor ablation, a T7 cancer gene therapy plasmid vector, pT7T7/T7TK, was constructed. This nonviral vector contains a T7 autogene, T7T7, and a human herpes simplex virus thymidine kinase (HSV-TK) gene driven by a second T7 promoter (T7TK). When co-transfected with T7 RNA polymerase (T7 RNAP) into cultured human osteosarcoma 143B cells, abut 10-20% of the cells were found to express HSV-TK, and more than 90% of the cells were killed in the presence of 1 microM ganciclovir (GCV) within 4 days after DNA transfection. The increase in killing above the transfection frequency is due to a "bystander" effect among transfected and untransfected 143B cells. Direct injections of pT7T7/T7TK into 143B tumors grown in nude mice resulted in TK gene expression in tumor cells located near the injection sites as revealed by the immunohistochemical staining. Repeated tumor injections of the pT7T7/T7TK vector and intraperitoneal (i.p.) injections of GCV resulted in inhibition of tumor growth and in tumor shrinkage in 6 out of 10 treated nude mice. Three of those six tumors fully regressed shortly after the end of the GCV injections. All of the full tumor regressions were found to be permanent and no apparent tumor relapses were observed for the rest of the lives of the treated nude mice after the initial tumor ablations. These results, combined with the nonviral and rapid cytoplasmic gene expression features, suggest that the T7 vector may be a good candidate for cancer gene therapy and other medical and biological applications.
Assuntos
Bacteriófago T7/genética , Neoplasias Ósseas/terapia , Terapia Genética , Vetores Genéticos , Osteossarcoma/terapia , Timidina Quinase/genética , Animais , RNA Polimerases Dirigidas por DNA/genética , Relação Dose-Resposta a Droga , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Herpes Simples/enzimologia , Humanos , Imuno-Histoquímica , Injeções Intraperitoneais , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
The regulation of gene expression by the tetracycline system has attracted a high level of interest in the recent past. However, expression of secreted proteins has not been evaluated precisely. In this study, we constructed two versions of a one-plasmid system containing the elements necessary for the regulation of gene expression. The regulatable elements and the selectable marker (Neor) were set up in two different configurations, pTRIN31 and pTRIN76. With these two regulatable versions, the levels of protein expression after transfection into the NIH/3T3 cell line were measured by insertion of three different genes encoding the secreted proteins (hGH, ApoE3, hGM-CSF). The maximum levels of gene expression obtained with the pTRIN76-derived plasmids were 100ng/24h/106 cells for hGH, 427ng/24h/106 cells for ApoE3 and 108ng/24h/106 cells for hGM-CSF. For the pTRIN31-derived plasmids the maximum levels were 2.7ng/24h/106 cells for hGH and 47ng/24h/106 for ApoE3. Both plasmids give rise to an expression of the transfected gene that can be tightly regulated by three different molecules: tetracycline, minocycline and doxycycline. The levels of the secreted proteins are below the detectable level when the reporter genes are repressed. This repression is reversible within 48h after the regulator has been removed from the medium.
Assuntos
Antibacterianos/farmacologia , Apolipoproteínas E/efeitos dos fármacos , Vetores Genéticos/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Hormônio do Crescimento Humano/efeitos dos fármacos , Células 3T3/citologia , Células 3T3/efeitos dos fármacos , Células 3T3/metabolismo , Animais , Apolipoproteína E3 , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Clonagem Molecular , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Camundongos , Minociclina/farmacologia , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Tetraciclina/farmacologiaRESUMO
The time course variations in tyrosine hydroxylase (TH) activity and specific mRNA were measured in the rat locus coeruleus (LC) and substantia nigra after an intracerebroventricular (i.c.v.) injection of 5,6-dihydroxytryptamine (5,6-DHT), a neurotoxin known to selectively destroy serotoninergic neurons. In this study, the TH activity and TH mRNA were both analyzed from homogenates of single tissue samples (micropunches). TH mRNA was extracted and quantified by densitometry using a northern blot method and an artificial TH RNA as an external standard. 5,6-DHT injection led to a long-lasting increase in TH activity and TH mRNA in LC but not in substantia nigra. The elevation in LC was progressive and reached its maximum value (+75%) at day 4 and day 8 after 5,6-DHT. This effect on TH activity was accompanied by a parallel change in TH mRNA whose amplitude was +57%, +81% and +45% at day 2, 4, and 8 respectively after the neurotoxin injection. Return to normal values was observed at day 16. Variations in TH activity and TH mRNA in LC were of similar amplitude. These results suggest that serotonin could be a potent modulator of TH gene expression within noradrenergic LC neurons.
Assuntos
5,6-Di-Hidroxitriptamina/farmacologia , Locus Cerúleo/efeitos dos fármacos , RNA Mensageiro/metabolismo , Tirosina 3-Mono-Oxigenase/genética , Animais , Injeções Intraventriculares , Locus Cerúleo/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
Alpha 1- and alpha 2-adrenoceptors were assessed by binding studies using [3H]prazosin and [3H]p-aminoclonidine as ligands in membrane preparations from the cortex, hippocampus and hypothalamus of rats, 3 weeks after intracerebroventricular injection of the neurotoxin 5,7-dihydroxytryptamine. Cortical alpha 1 and hippocampal alpha 2 adrenoceptors were significantly increased. Treatment also affected the affinity of cortical alpha 2 adrenoceptors. These results suggest a heterologous, region-specific regulation of both subtypes of central alpha-adrenergic receptors by serotonin.
Assuntos
Encéfalo/fisiologia , Receptores Adrenérgicos alfa/fisiologia , Serotonina/fisiologia , 5,7-Di-Hidroxitriptamina/farmacologia , Animais , Córtex Cerebral/fisiologia , Clonidina/análogos & derivados , Clonidina/metabolismo , Hipocampo/fisiologia , Hipotálamo/fisiologia , Masculino , Prazosina/metabolismo , Ratos , Ratos EndogâmicosRESUMO
BACKGROUND: An anti-angiogenesis strategy has been widely recognized as a viable approach to fight cancer and more and more anti-angiogenic factors are continually being identified. Among them, the muscular isoform of Troponin I (TnI) has been described as being a powerful anti-angiogenic agent in vitro as well as in vivo. We investigated the therapeutic efficacy of TnI gene therapy in a human-like orthotopic rat osteosarcoma model. MATERIALS AND METHODS: In this tumor model, we evaluated whether the administration of the secreted TnI coding sequence complexed to cationic liposomes (named TnITag cDNA/lCLP) could induce a delay in tumor growth and reduce tumor vasculature. RESULTS: Although TnI specifically inhibited endothelial cell growth in vitro, we were not able to demonstrate any therapeutic efficacy of TnI in the transplantable osteosarcoma model. CONCLUSION: This lack of efficacy probably resulted from the rapid degradation of recombinant TnI by matrix metalloproteinases, especially MMP2, which are present in large amounts in tumors.
Assuntos
Terapia Genética/métodos , Osteossarcoma/irrigação sanguínea , Osteossarcoma/terapia , Troponina I/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , DNA Complementar/administração & dosagem , DNA Complementar/genética , Modelos Animais de Doenças , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Lipossomos/administração & dosagem , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , Osteossarcoma/genética , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , TransfecçãoRESUMO
The use of DNA vectors to elicit an immune response has produced a lot of interest. Unfortunately, one of the limiting factors has been the problem of gene expression. In order to obtain a strong expression of the vaccinating gene, several steps are necessary. The vector has to be delivered in such a way that it is not being degraded by the immune nor by the hepatic system; it has also to enter efficiently the targeted cells; and it must be expressed in the appropriate compartment of the cells at a high level. For these reasons, we have developed a gene expression vector that contains a T7 autogene and is being expressed in the cytoplasm of the cells (1,2). We will describe this system and two possible applications: infectious disease vaccination and tumor ablation. The latter application may be combined with DNA vaccination against cancer cells.
RESUMO
The tetracycline family is composed of several molecules whose antibacterial properties are due to the fixation on the bacterial ribosomes. Among those, doxycycline is one of the most potent antibiotics for which additional features have been recently discovered. Doxycycline has been found to inhibit metalloproteinases, to decrease gelatinolytic and metastatic activities of cancer cells, to have a "chondroprotective" effect in inflammatory arthritides, and to have strong antimalarial properties. In this study, a murine retrovirus producing cell line (psi CRIP-pXT1) was incubated in variable concentrations of doxycycline. The retroviral titer of this cell line was measured by the ability to transfer resistance to G418 to NIH/3T3 cells. The retroviral titer was significantly decreased by 70% when the packaging cells had been incubated with 25 microM of doxycycline at 37 degrees C. The ID50 was around 8 micrograms/ml. Astonishingly, this effect was not observed at 32 degrees C. The mechanism of this effect is still to be determined. It may be useful to be aware of this effect for uncovering all of the possible antiviral qualities of doxycycline and its related molecules, such as glycylcyclines or anthracyclines.
Assuntos
Antivirais/farmacologia , Doxiciclina/farmacologia , Vírus da Leucemia Murina de Moloney/efeitos dos fármacos , Retroviridae/efeitos dos fármacos , Células 3T3 , Animais , Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Técnicas de Transferência de Genes , Camundongos , TemperaturaRESUMO
Charcot-Marie-Tooth disease (Hereditary Motor and Sensory Neuropathy) sometimes begins during childhood and can lead to learning and/or orthopedic disabilities. Due to the genetic and clinical heterogeneity of the disease, the diagnosis is based on a familial study of clinical, electromyographic and pathological abnormalities. Two major types of Charcot-Marie-Tooth disease have been described. Type 1 is characterized by a decrease in nerve conduction velocities and by a peripheral nerve hypertrophy due to myelinic alterations, while type 2 is the consequence of axonal alterations. Although type 1 and type 2 patients share similar clinical symptoms, type 2 patients have normal nerve conduction velocities and histological signs of axonal damage. Several genes involved in this disease have been recently located, and, in certain cases, an individual and direct diagnosis is available if the familial abnormality is related to chromosome 17.
Assuntos
Doença de Charcot-Marie-Tooth , Adolescente , Doença de Charcot-Marie-Tooth/classificação , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/genética , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Gravidez , PrognósticoRESUMO
Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy first described in 1886. Our increasing knowledge of this disease correlates well with the development of methods used in neurology over the past 100 years. Although its physiopathology and treatment is still not fully understood, current developments in techniques are opening the way to future discoveries. We have divided its history into three theoretical periods: the first from 1886 to 1956, which was devoted to clinical and pathological study of the disease; the second from 1956 to 1982, which saw the development of electromyography in the investigation of neuromuscular diseases; and the last and current period based upon genetic research, using the methods of molecular biology.
Assuntos
Doença de Charcot-Marie-Tooth/história , Doenças Genéticas Inatas/história , França , História do Século XIX , História do Século XX , Humanos , Reino UnidoRESUMO
The "medical biologist" has to play a role as a consultant for the relevant use of biological examinations and to provide comments for their interpretation, comprehensible and useful to physicians. Advisory activities of the medical laboratory may help physician in diagnosis or therapeutic algorithm, avoiding ordering incomplete or useless examinations. After presentation of regulation and requirements of the EN ISO 15189 standard, this paper gives proposals for recommendations to apply in the context of accreditation. A proven and regular continuing education program is needed and professional practices must be supported by recognized and recent guidelines. This document provides suggestions for advisory services traceability and reports a list of websites and articles to use in defining standardised comments as well.
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OBJECTIVE: Severe early-onset axonal neuropathy (SEOAN) is a heterogeneous phenotype first delineated by Ouvrier et al., characterized by progressive axonal degeneration with gait problems often progressing to wheelchair requirement and later respiratory involvement. Most cases are sporadic single cases. Some have heterozygous mitofusin 2 (MFN2) mutations, many of which are de novo dominant mutations. The aim of this study was to investigate the mode of inheritance in three individuals with severe early-onset axonal neuropathy and homozygous or compound heterozygous MFN2 mutations. METHODS: The clinical and molecular findings in the parents of three individuals with SEOAN with homozygous or compound heterozygous MFN2 mutations were examined. RESULTS: All parents were asymptomatic or mildly symptomatic with some signs of peripheral neuropathy indicating a minimal phenotype. Two had hearing problems. All parents carried the relevant single base (heterozygous) MFN2 variations. CONCLUSION: Severe early-onset axonal neuropathy due to MFN2 mutations can present as an apparently recessively inherited neuropathy but the minimal phenotype in the parents suggests a semi-dominant mechanism.
Assuntos
Axônios/metabolismo , Predisposição Genética para Doença/genética , Heterozigoto , Homozigoto , Proteínas de Membrana/genética , Proteínas Mitocondriais/genética , Mutação/genética , Doenças do Sistema Nervoso Periférico/genética , Adulto , Idade de Início , Axônios/patologia , Análise Mutacional de DNA , Feminino , GTP Fosfo-Hidrolases , Genes Dominantes/genética , Marcadores Genéticos/genética , Testes Genéticos , Genótipo , Perda Auditiva Neurossensorial/genética , Humanos , Padrões de Herança/genética , Masculino , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Nervos Periféricos/fisiopatologia , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/fisiopatologia , Degeneração Walleriana/genética , Degeneração Walleriana/metabolismo , Degeneração Walleriana/fisiopatologiaRESUMO
The gene therapy strategy using the hsv1-thymidine kinase gene (TK) and ganciclovir (GCV) injections that has been used for treating human glioblastomas has not been as effective as expected after the first animal experiments. A better understanding of the different steps involved in this treatment, like gene transfer, gene expression, and sensitivity of the recipient cells is needed. Therefore, we studied 7 human glioblastoma cell lines (U87, U118, U251, SNB19, SNB75, SF295, SF539) for their sensitivity to the TK/GCV system. We also studied their in vitro bystander effect and their in vitro transfectability using LipofectAMINE as a transfection enhancer. According to this in vitro analysis, most of the glioblastoma cell lines should be sensitive to the TK/GCV system, but there is a significant need for agents to increase transfection efficiency.
Assuntos
Ganciclovir/uso terapêutico , Terapia Genética , Glioblastoma/terapia , Animais , Expressão Gênica , Técnicas de Transferência de Genes , Glioblastoma/patologia , Humanos , Ratos , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Transfecção , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/patologiaRESUMO
In some countries with a high prevalence of consanguineous marriages, autosomal recessive inheritance is likely to account for the great majority of all forms of Charcot-Marie-Tooth (CMT) disease. As with the dominant forms, it is usual to differentiate the demyelinating forms (autosomal recessive -CMT1 or AR-CMT4) from the axonal forms (AR-CMT2). Genetic analysis of large families with recessive transmission has proved to be an efficient mean of discovering novel CMT genotypes (eg, the genes GDAP1, MTMR2, MTMR13, KIAA1985, NDGR1, periaxin, and lamin). Because of the clinical, electrophysiologic, and histologic heterogeneity of these patients, it is likely that there are numerous genes that remain to be discovered, which will probably make classification even more complex. Clinical, and especially histologic, phenotypes often lead to a suspicion that a specific gene is implicated. There is, therefore, an indication for nerve biopsy to orient diagnostic research in molecular biology, which is presently very time consuming and can only be performed in highly specialized laboratories.