RESUMO
The ability of a replication-deficient adenovirus vector to transfer a foreign gene into neural cells of adult rats in vivo has been analysed. A large number of neural cells (including neurons, astrocytes and ependymal cells) expressed an E. coli lacZ transgene for at least 45 days after inoculation of various brain areas. Injecting up to 3 x 10(5) pfu in 10 microliters did not result in any detectable cytopathic effects--these were only observed for very high titres of infection (> 10(7) pfu 10 microliters-1). Adenovirus vectors therefore appear to be a promising means for in vivo transfer of therapeutic genes into the central nervous system.
Assuntos
Adenoviridae/genética , Encéfalo/citologia , Genes Bacterianos , Neurônios/citologia , Transfecção/métodos , beta-Galactosidase/metabolismo , Animais , Encéfalo/enzimologia , Escherichia coli/enzimologia , Escherichia coli/genética , Terapia Genética/métodos , Vetores Genéticos , Complexo de Golgi/enzimologia , Complexo de Golgi/ultraestrutura , Neurônios/enzimologia , Ratos , Ratos Sprague-Dawley , Recombinação Genética , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy cause progressive paralysis, often leading to premature death. Neurotrophic factors have been suggested as therapeutic agents for motor neuron diseases, but their clinical use as injected recombinant protein was limited by toxicity and/or poor bioavailability. We demonstrate here that adenovirus-mediated gene transfer of neurotrophin-3 (NT-3) can produce substantial therapeutic effects in the mouse mutant pmn (progressive motor neuronopathy). After intramuscular injection of the NT-3 adenoviral vector, pmn mice showed a 50% increase in life span, reduced loss of motor axons and improved neuromuscular function as assessed by electromyography. These results were further improved by coinjecting an adenoviral vector coding for ciliary neurotrophic factor. Therefore, adenovirus-mediated gene transfer of neurotrophic factors offers new prospects for the treatment of motor neuron diseases.
Assuntos
Terapia Genética/métodos , Doença dos Neurônios Motores/terapia , Fatores de Crescimento Neural/uso terapêutico , Proteínas do Tecido Nervoso/uso terapêutico , Adenoviridae/genética , Animais , Animais Recém-Nascidos , Fator Neurotrófico Ciliar , Eletromiografia , Vetores Genéticos , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Mutantes , Doença dos Neurônios Motores/mortalidade , Músculos/inervação , Degeneração Neural/efeitos dos fármacos , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Neurotrofina 3 , Nervo Frênico/patologia , Análise de SobrevidaRESUMO
We have previously demonstrated that there is a low level of transcription of tissue-specific genes in every cell type. In this study, we have taken advantage of this phenomenon, called illegitimate transcription, to analyze the muscle-type dystrophin mRNA in easily accessible cells such as lymphoid cells, fibroblasts, and peripheral blood cells from Duchenne and Becker muscular dystrophies with known internal gene deletion. The results showed that, in the studied regions surrounding the deletions, processing of truncated transcripts is identical in specific (muscle tissue) and in nonspecific cells (lymphoid cells). In Becker cases with out-of-frame deletions, the already described alternatively spliced species found in muscle samples were also found in nonspecific cells. These results demonstrate that illegitimate transcripts are a bona fide version of tissue-specific mRNA, and that they represent a useful material to investigate the qualitative consequences of gene defects at the mRNA level.
Assuntos
Distrofina/genética , Distrofias Musculares/genética , Transcrição Gênica , Sequência de Bases , Células Cultivadas , Humanos , Dados de Sequência Molecular , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Splicing de RNA , RNA Mensageiro/análiseRESUMO
The serine protease inhibitor, elafin, is a critical component of the epithelial barrier against neutrophil elastase (NE). Elafin is downregulated in the majority of breast cancer cell lines compared with normal human mammary epithelial cells (HMECs). Here, we evaluated the role of elafin and NE on proliferation and tumorigenesis. Elafin is induced in growth factor-deprived HMECs as they enter a quiescent (G0) state, suggesting that elafin is a counterbalance against the mitogenic effects of NE in G0 HMECs. Stable knockdown of elafin compromises the ability of HMECs to maintain G0 arrest during long-term growth factor deprivation; this effect can be reversed by re-expression of wild-type elafin but not elafin-M25G lacking protease inhibitory function. These results suggest that NE, which is largely contributed by activated neutrophils in the tumor microenvironment, may be negatively regulating the ability of elafin to arrest cells in G0. In fact when purified NE was added to elafin-knocked down HMECs, these cells demonstrated greater sensitivity to the growth-promoting effects of purified NE. Activation of ERK signaling, downstream of toll-like receptor 4, was essential to the mitogenic effect of NE on HMECs. These findings were next translated to patient samples. Immunohistochemical analysis of normal breast tissue revealed robust elafin expression in the mammary epithelium; however, elafin expression was dramatically downregulated in a significant proportion of human breast tumor specimens. The loss of elafin expression during breast cancer progression may promote tumor growth as a consequence of increased NE activity. To address the role of NE in mammary tumorigenesis, we next examined whether deregulated NE activity enhances mammary tumor growth. NE knockout in the C3(1)TAg mouse model of mammary tumorigenesis suppressed proliferation and reduced the kinetics of tumor growth. Overall, the imbalance between NE and its inhibitors, such as elafin, presents an important therapeutic target in breast cancer.
Assuntos
Proliferação de Células/genética , Elafina/fisiologia , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Elafina/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Elastase de Leucócito/farmacologia , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitose/efeitos dos fármacos , Mitose/genéticaRESUMO
Tay-Sachs disease is a lipidosis due to the deficiency of the lysosomal hexosaminidase A. In order to understand the molecular mechanisms of this enzyme deficiency we studied 42 patients of different ethnic origins diagnosed in Europe. The strategy used consists in HEXA cDNA amplification followed by allele-specific oligonucleotide analysis for the frequent mutations, and by chemical cleavage mismatch and denaturing gradient gel electrophoresis for the detection of new mutations. 90% of alleles were clarified in this way, showing a high heterogeneity of HEXA lesions in Tay-Sachs disease. 28 different mutations were found, 20 being identified for the first time in this group of patients.
Assuntos
Mutação , Doença de Tay-Sachs/epidemiologia , Doença de Tay-Sachs/genética , beta-N-Acetil-Hexosaminidases/genética , Adulto , Alelos , Composição de Bases , Sequência de Bases , Pré-Escolar , Análise Mutacional de DNA/métodos , Fosfatos de Dinucleosídeos/genética , Eletroforese em Gel de Poliacrilamida/métodos , Mutação da Fase de Leitura , Genótipo , Hexosaminidase A , Humanos , Lactente , Epidemiologia Molecular , Dados de Sequência Molecular , Mutagênese Insercional , Ácidos Nucleicos Heteroduplexes/genética , Sondas de Oligonucleotídeos , Fenótipo , Mutação Puntual , Splicing de RNA , Deleção de SequênciaRESUMO
Adenovirus vectors containing a marker gene (lacZ from Escherichia coli) are potent for transferring the gene to neurones after intraparenchymal injections. Expression of the marker gene may lead to the synthesis of an enormous amount of beta-galactosidase which diffuses throughout the entire neurone, providing a 'Golgi-like' staining. This suggested that the technique may be used to study the morphology of specific neuronal populations. We have validated this hypothesis by analysing the postnatal development of motoneurones in the rat cervical cord. Injections of the viral suspension into one ventral horn were performed at different ages after birth. Histochemical staining using X-Gal revealed morphological changes occurring within the first 3 weeks with enlargement of the perikaryon and increased dendritic complexity. Immunoreactivity for CGRP was visualized in double-staining experiments. In vivo transfer of a marker gene therefore provides a new way to analyse neuronal morphology which allows selection of the cells to be studied and double-labelling with immunohistochemical markers.
Assuntos
Técnicas de Transferência de Genes , Neurônios Motores/fisiologia , Medula Espinal/crescimento & desenvolvimento , Adenoviridae/genética , Animais , Peptídeo Relacionado com Gene de Calcitonina/imunologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Dendritos/ultraestrutura , Marcadores Genéticos/imunologia , Vetores Genéticos , Imuno-Histoquímica , Ratos , Ratos Sprague-Dawley , Medula Espinal/citologia , Medula Espinal/enzimologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
The abnormalities in the gene coding for the beta-hexosaminidase alpha subunit were analysed from fibroblast's RNAs of 42 Tay-Sachs patients (seven with adult or late onset of Tay-Sachs disease and 35 with infantile Tay-Sachs disease). After first strand synthesis by random priming, PCR was used to amplify in two overlapping fragments (868 and 949 bp) the entire coding region. These amplified products were first studied for changes in size by agarose gel electrophoresis to screen for splicing mutations leading to exon skipping or cryptic splice site activation. For each patient, the two overlapping cDNA fragments were subjected to chemical mismatch cleavage analysis using hydroxylamine to modify C-containing mismatches and osmium tetroxide to modify T-containing mismatches. DGGE was used to screen for mutations in the coding region spanning exon 2 to exon 6, a region putatively encompassing the active site and therefore a potential hot spot of mutations associated with Tay-Sachs disease. To increase the sensitivity of the technique, a 30 bp GC-clamp has been added at the 5' end of the sense oligonucleotide to amplify a fragment of 629 bp. The computerized analysis found that single base changes in domain spanning from nt 313 to nt 693 can be distinguished. Fragments displaying an altered melting behavior or a cleaved product were further analysed by direct sequencing of the amplified material. These methods as a whole allowed us to identify 30/38 alleles studied (79%) with 15 point mutations and one 4 bp insertion detected.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Doença de Tay-Sachs/epidemiologia , Doença de Tay-Sachs/genética , Adulto , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Europa (Continente)/epidemiologia , Genótipo , Humanos , Lactente , Mutação , Reação em Cadeia da Polimerase , Doença de Tay-Sachs/etnologiaRESUMO
HER2/neu (HER2) and cyclin E are important prognostic indicators in breast cancer. As both are involved in cell cycle regulation we analyzed whether there was a direct interaction between the two. HER2 and cyclin E expression levels were determined in 395 breast cancer patients. Patients with HER2-overexpression and high levels of cyclin E had decreased 5-year disease-specific survival compared with low levels of cyclin E (14% versus 89%, P<0.0001). In vitro studies were performed in which HER2-mediated activity in HER2-overexpressing breast cancer cell lines was downregulated by transfection with HER2 small interfering RNA or treatment with trastuzumab. Cyclin E expression levels were determined by western blot analysis, and functional effects analyzed using kinase assays, MTT assays were used to assess cell viability as a marker of proliferation and fluorescence-activated cell sorting analysis was used to determine cell cycle profiles. Decreased HER2-mediated signaling resulted in decreased expression of cyclin E, particularly the low molecular weight (LMW) isoforms. Decreased HER2 and LMW cyclin E expression had functional consequences, including decreased cyclin E-associated kinase activity and decreased proliferation, because of increased apoptosis and an increased accumulation of cells in the G1 phase. In vivo studies performed in a HER2-overexpressing breast cancer xenograft model confirmed the effects of trastuzumab on cyclin E expression. Given the relationship between HER2 and cyclin E, in vitro clonogenic assays were performed to assess combination therapy targeting both proteins. Isobologram analysis showed a synergistic interaction between the two agents (trastuzumab targeting HER2 and roscovitine targeting cyclin E). Taken together, these studies show that HER2-mediated signaling effects LMW cyclin E expression, which in turn deregulates the cell cycle. LMW cyclin E has prognostic and predictive roles in HER2-overexpressing breast cancer, warranting further study of its potential as a therapeutic target.
Assuntos
Neoplasias da Mama/metabolismo , Ciclina E/metabolismo , Receptor ErbB-2/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina E/química , Ciclina E/genética , Regulação para Baixo , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Peso Molecular , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Taxa de Sobrevida , Transcrição Gênica , TrastuzumabRESUMO
E1A can evoke G1 exit in cardiac myocytes and other cell types by displacing E2F transcription factors from tumor suppressor "pocket" proteins and by a less well-characterized p300-dependent pathway. Bypassing pocket proteins (through overexpression of E2F-1) reproduces the effect of inactivating pocket proteins (through E1A binding); however, pocket proteins associate with a number of molecular targets apart from E2F. Hence, pocket protein binding by E1A might engage mechanisms for cell cycle reentry beyond those induced by E2F-1. To test this hypothesis, we used adenoviral gene transfer to express various E2F-1 and E1A proteins in neonatal rat cardiac myocytes that are already refractory to mitogenic serum, in the absence or presence of several complementary cell cycle inhibitors-p16, p21, or dominant-negative cyclin-dependent kinase-2 (Cdk2). Rb binding by E2F-1 was neither necessary nor sufficient for G1 exit, whereas DNA binding was required; thus, exogenous E2F-1 did not merely function by competing for the Rb "pocket." E2F-1-induced G1 exit was blocked by the "universal" Cdk inhibitor p21 but not by p16, a specific inhibitor of Cdk4/6; p21 was permissive for E2F-1 induction of cyclins E and A, but prevented their stimulation of Cdk2 kinase activity. In addition, E2F-1-induced G1 exit was blocked by dominant-negative Cdk2. Forced expression of cyclin E induced endogenous Cdk2 activity but not G1 exit. Thus, E2F-1-induced Cdk2 function was necessary, although not sufficient, to trigger DNA synthesis in cardiac muscle cells. In contrast, pocket protein-binding forms of E1A induced G1 exit that was resistant to inhibition by p21, whereas G1 exit via the E1A p300 pathway was sensitive to inhibition by p21. Both E1A pathways-via pocket proteins and via p300-upregulated cyclins E and A and Cdk2 activity, consistent with a role for Cdk2 in G1 exit induced by E1A. However, p21 blocked Cdk2 kinase activity induced by both E1A pathways equally. Thus, E1A can cause G1 exit without an increase in Cdk2 activity, if the pocket protein-binding domain is intact. E1A also overrides p21 in U2OS cells, provided the pocket protein-binding domain is intact; thus, this novel function of E1A is not exclusive to cardiac muscle cells. In summary, E1A binding to pocket proteins has effects beyond those produced by E2F-1 alone and can drive S-phase entry that is resistant to p21 and independent of an increase in Cdk2 function. This suggests the potential involvement of other endogenous Rb-binding proteins or of alternative E1A targets.
Assuntos
Proteínas E1A de Adenovirus/fisiologia , Quinases relacionadas a CDC2 e CDC28 , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Ciclinas/fisiologia , Miocárdio/citologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Células Cultivadas , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Ativação Enzimática , Regulação da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Coração/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
The molecular basis of null alleles was investigated by cDNA polymerase chain reaction (PCR) in seven Tay-Sachs patients. Although mRNAs were undetectable by Northern blot, cDNA-PCR amplification allowed us to get a sufficient amount of cDNA to characterize abnormal transcripts. In two French patients (one homozygote and one compound heterozygote with a 4-bp insertion in exon 11 of the second allele) suffering an infantile form of the disease, we detected abnormal RNAs with a 17-bp insertion due to a GT to AT transition at the donor site of intron 9, resulting in the activation of a cryptic donor site in the intron. This mutation has been found in 9 out of 82 Tay-Sachs chromosomes (11%) in association with alleles responsible from different clinical courses. In the other five patients we found the 4-bp insertion in exon 11 and two nonsense mutations.
Assuntos
Alelos , Doença de Tay-Sachs/genética , Sequência de Bases , Northern Blotting , Análise Mutacional de DNA , Éxons , Feminino , Mutação da Fase de Leitura , Humanos , Lactente , Íntrons , Masculino , Dados de Sequência Molecular , Linhagem , Mutação Puntual , Reação em Cadeia da Polimerase , Splicing de RNA , RNA Mensageiro/análiseRESUMO
In hereditary neurological diseases, gene transfer into neurons is made difficult by: the nature of the cells (postmitotic cells, that cannot be cultured, genetically modified ex vivo, then retransplanted), sometimes, their widespread localization, the blood-brain barrier. However, three viral vectors derived from adenovirus, Herpes simplex virus and adeno-associated virus have been shown to be very efficient in transferring DNA into brain cells. All of these vectors can infect resting cells, especially neurons, and are efficient in vivo. Retroviral vectors which can infect dividing cells only are mainly used for ex vivo genetic modification of cells (neural progenitor cells, myoblasts, fibroblasts) followed by intracerebral transplantation. Alternatively, genetically modified cells can be transplanted in a peripheral site if the transgene product is able to cross the blood-brain barrier or to be transported retrogradely from the nerve terminals. We have especially investigated the potential interest of adenoviral vectors to transfer foreign genes into brain cells and to treat animal models of neurological diseases. These vectors allowed us to transfer the lacZ gene into any neural cell type, including neurons, glia, photoreceptors and olfactory receptors, ex vivo, in cell culture, and in vivo, by stereotactic administration. In addition, axonal transport of adenoviral vectors has been demonstrated, e.g. in the substantia nigra after injection into the striatum, in the olfactory bulb after intranasal instillation and in spinal motor neurons after intramuscular injection. After intracerebroventricular injection, ependymal cells are massively infected and express the transgene for several months, as this is also observed in neurons. Through the spinal canal and cerebrospinal fluid, the vector can diffuse to a considerable distance from the injection point, e.g. to the lumbar spinal cord after injection in the suboccipital region. To test the biological function of transgenes transferred through adenoviral vectors, we have constructed vectors with cDNAs or genes for various neutrophic factors: CNTF, NT3, BDNF and GDNF. These vectors were biologically active on target cells, ex vivo and in vivo. In the pmn mouse model of progressive motor neuronal degeneration, some of these vectors, alone or combined, allowed for prolongation of life of homozygous animals by more than two fold, and for decrease in the demyelination of phrenic nerve axons. Finally, we have also constructed an adenoviral vector carrying the alpha-hexosaminidase cDNA, encoding the enzyme subunit deficient in Tay Sachs patients. This vector permitted to normalize ganglioside metabolism in Tay Sachs fibroblasts and is currently tested in knock out mice deficient in hexosaminidase A. In spite of all these encouraging results, we are nevertheless aware that progress in vector design and delivery strategies will be needed before gene therapy can become a realistic therapeutical strategy in humans.
Assuntos
Terapia Genética , Neuropatias Hereditárias Sensoriais e Autônomas/terapia , Adenoviridae/genética , Animais , Vetores GenéticosRESUMO
Total RNA was isolated from cultured fibroblasts from 12 unrelated patients with Tay-Sachs disease, an autosomal recessive disorder due to beta-hexosaminidase A deficiency. beta-Hexosaminidase mRNA was amplified by cDNA-PCR in four overlapping segments spanning the entire coding sequence. In two patients, abnormal size cDNA-PCR fragments in which exons were removed resulted from splicing mutations that were characterized at the genomic DNA level: both were G to A transitions, at the first position of intron 2 and at the fifth position of intron 4. Five other mutations have been identified by cDNA-PCR chemical mismatch analysis and direct sequencing of an amplified fragment containing the mismatch site. One missense mutation alters the codon for Ser210 to Phe in exon 6 and the other one alters the codon for Arg504 to Cys in exon 13. A 3-bp deletion results in the deletion of a phenylalanine residue in exon 8. Two nonsense mutations in exon 3 (Arg137 to stop) and in exon 11 (Arg393 to stop) are associated with a marked decrease of mRNA abundance, probably because they result in mRNA instability. Three of the six single base mutations involve the conversion of a CpG dinucleotide in the sense strand to TpG. These results demonstrate the extreme molecular heterogeneity of mutations causing Tay-Sachs disease. The procedure described in this paper allows the rapid detection of any type of mutation, except those impairing the promoter function. Applicable even to patients with splicing or nonsense mutations and very low mRNA abundance, it has therefore a potentially broad application in human genetics, for both diagnostic and fundamental purposes.
Assuntos
Mutação , Doença de Tay-Sachs/genética , Sequência de Bases , Linhagem Celular , DNA , Análise Mutacional de DNA , Eletroforese em Gel de Ágar , Técnicas Genéticas , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Doença de Tay-Sachs/etnologia , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
Prenatal diagnosis of metachromatic leukodystrophy (MLD) due to arylsulphatase A (ASA) deficiency can be performed by amniocentesis with the disadvantage of a late pregnancy termination. Whether chorionic villi (CV) obtained by trophoblast biopsy during the first trimester of pregnancy can be useful for diagnosis depends on the reliability of results. The complexity of arylsulphatase expression in CV and the existence of several isozymes make diagnosis difficult. However, the use of an anti-ASA antiserum enabled us to discriminate between ASA and a comigrating contaminant isozyme, and the antigen-antibody (Ag-Ab) complex gave better evidence of the presence or absence of ASA after enhancement of activity with 4-methylumbelliferyl sulphate (4-MUS). We propose that immunoprecipitation followed by electrophoresis could be a valuable method of MLD prenatal diagnosis on chorionic villi.
Assuntos
Cerebrosídeo Sulfatase/análise , Amostra da Vilosidade Coriônica/métodos , Leucodistrofia Metacromática/diagnóstico , Diagnóstico Pré-Natal , Precipitação Química , Feminino , Humanos , Imunoeletroforese , Gravidez , Primeiro Trimestre da GravidezRESUMO
Beta-mannosidosis is a recently described inherited disorder with predominantly neurological signs and symptoms as the major manifestations of the disorder. The heterogeneous manifestations of the disease have been presented in seven previous patients. We describe a further case of European descent with an infantile onset of the disease, with the features of speech impairment as the first symptom. Beta-mannosidase activity was completely deficient in the patient and a heterozygote level was found in the parents. In addition, mannosyl-N-acetylglucosamine was identified in the patient's urine in keeping with the diagnosis of beta-mannosidosis.
Assuntos
Sintomas Afetivos/etiologia , Manosidases/deficiência , Distúrbios da Fala/etiologia , alfa-Manosidose/psicologia , Pré-Escolar , Humanos , Masculino , Oligossacarídeos/urina , alfa-Manosidose/complicações , alfa-Manosidose/enzimologia , beta-ManosidaseRESUMO
We investigated the possibility of prenatal diagnosis of mucolipidosis type II (ML II) by lysosomal enzyme determination on amniotic fluid obtained at 11 weeks of gestation in three pregnancies at risk. Diagnosis of ML II was made in one case on the basis of increased levels of five lysosomal enzymes tested. The diagnosis was confirmed on cultured chorionic cells, their cultured medium, 17-week amniotic fluid, and fetal plasma obtained for confirmation prior to the termination of pregnancy.
Assuntos
Amniocentese , Líquido Amniótico/análise , Hidrolases/análise , Mucolipidoses/diagnóstico , Adulto , Cerebrosídeo Sulfatase/análise , Condro-4-Sulfatase/análise , Consanguinidade , Feminino , Glucuronidase/análise , Hexosaminidases/análise , Humanos , Manosidases/análise , Gravidez , Primeiro Trimestre da Gravidez , alfa-L-Fucosidase/análise , alfa-Manosidase , beta-Galactosidase/análiseRESUMO
Tay-Sachs disease is an autosomal recessive genetic disease caused by a deficiency in beta-hexosaminidase A. We have characterized a new mutation in a Tunisian patient displaying a late infantile form of Tay-Sachs disease. Northern blot analysis of patient's fibroblast total RNAs shows a broad, fast migrating band in the region of the normal beta-hexosaminidase alpha transcripts. The mRNA coding for beta-hexosaminidase alpha subunit was first reverse transcribed and then amplified in four overlapping segments spanning the entire coding sequence by polymerase chain reaction. We found in the products of polymerase chain reaction (PCR) that amplify the segment spanning exons 2-7, in addition to a normal fragment, two smaller size fragments, one of which is also seen in normal control fibroblasts. The analysis of the patient's specific abnormal fragment by hybridization with exon-specific oligonucleotides and then sequencing allowed us to conclude that this fragment lacked exon 5. The other smaller species lacked exons 4 and 5 in both patient and normal control. The sequence of a genomic fragment containing exon 5 and of the patient's normal cDNA fragment spanning exons 2-7, revealed a point mutation G to A at the last nucleotide of exon 5. This mutation doesn't change the sense of the affected codon. Northern blot of patient's fibroblast poly(A+) RNAs allowed us to quantify two of the forms of transcripts seen by PCR. In the patient, the normal size transcript and the exon 5-deleted transcript represent, respectively, 3 and 7% of the normal control beta-hexosaminidase alpha mRNA. We propose that this point mutation is responsible for an inefficient and abnormal processing of the mutant transcript resulting in the appearance of two low abundance spliced mRNAs. One is lacking exon 5 and most likely codes for an inactive protein; the other is similar to normal beta-hexosaminidase alpha mRNA, except for the presence of the silent G to A mutation and codes therefore for a normal enzyme accounting for the 2.5% residual beta-hexosaminidase A activity measured in patient's fibroblasts by a fluorometric assay. The third form, without exons 4 and 5, is also evidenced in normal fibroblasts by PCR so that we think that it is not related to Tay-Sachs disease.
Assuntos
Adenina , Guanina , Mutação , Splicing de RNA , Doença de Tay-Sachs/genética , Sequência de Bases , Northern Blotting , Células Cultivadas , DNA/genética , Éxons , Fibroblastos/enzimologia , Humanos , Lactente , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Transcrição Gênica , beta-N-Acetil-Hexosaminidases/genéticaRESUMO
Tay-Sachs disease is a severe neurodegenerative disorder due to mutations in the HEXA gene coding for the alpha-chain of the alpha-beta heterodimeric lysosomal enzyme beta-hexosaminidase A (HexA). Because no treatment is available for this disease, we have investigated the possibility of enzymatic correction of HexA-deficient cells by HEXA gene transfer. Human HEXA cDNA was subcloned into a retroviral plasmid generating to G.HEXA vector. The best Psi-CRIP producer clone of G.HEXA retroviral particles was isolated, and murine HexA-deficient fibroblasts derived from hexa -/- mice were transduced with the G.HEXA vector. Transduced cells overexpressed the alpha-chain, resulting in the synthesis of interspecific HexA (human alpha-chain/murine beta-chain) and in a total correction of HexA deficiency. The alpha-chain was secreted in the culture medium and taken up by HexA-deficient cells via mannose-6-phosphate receptor binding, allowing for the restoration of intracellular HexA activity in non-transduced cells.
Assuntos
Doença de Tay-Sachs/enzimologia , Doença de Tay-Sachs/genética , Transfecção , beta-N-Acetil-Hexosaminidases/genética , Células 3T3 , Animais , Clonagem Molecular , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hexosaminidase A , Humanos , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Retroviridae/genética , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
The heterogeneity of mutations causing Tay-Sachs disease in non-Jewish populations requires efficient techniques allowing the simultaneous screening for both known and novel mutations. beta-hexosaminidase mRNA isolated from cultured fibroblasts of 19 Tay-Sachs patients (7 with adult or late onset form of the disease and 12 with infantile Tay-Sachs disease) was amplified by cDNA-PCR in two overlapping segments spanning the entire coding sequence. We used chemical mismatch cleavage (CMC), denaturing gradient gel electrophoresis (DGGE) and direct sequencing of amplified fragments displaying a cleaved product or an altered melting behavior to screen the HEX A gene for mutations and to determine their distribution and frequency in the non-Jewish Tay-Sachs patients. These methods allowed us to identify 31 out of 38 alleles studied (82%). In addition to 9 previously described mutations (the 4 bp insertion in exon 11, G to A transitions at codons 170, 269, 482, 499 and 504, C to T transition at codon 499 and 504 and a GT to AT transition at the donor site of intron 9), we have identified 10 novel mutations. These include 1 donor splice site defect in intron 6, 8 missense mutations at non-randomly distributed conserved residues and a 2 bp deletion in exon 4. These results confirm the extreme molecular heterogeneity of mutations causing Tay-Sachs disease in non-Jewish population. The strategy used should be profitable for identifying mutations in large genes and for diagnostic purposes.
Assuntos
Mutação Puntual , Doença de Tay-Sachs/enzimologia , Doença de Tay-Sachs/genética , beta-N-Acetil-Hexosaminidases/genética , Adulto , Sequência de Aminoácidos , Sequência de Bases , Células Cultivadas , Etnicidade , Éxons , Fibroblastos/enzimologia , Frequência do Gene , Hexosaminidase A , Humanos , Lactente , Íntrons , Judeus , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificaçãoRESUMO
Previous studies have demonstrated that a defective recombinant adenovirus can infect a wide range of postmitotic and slowly proliferating cell types such as hepatocytes, myotubes, pneumocytes and intestinal cells (Stratford-Perricaudet et al., Hum. Gene Ther., 1, 241-256, 1990; Quantin et al., Proc. Natl. Acad. Sci. USA, 89, 2581-2584, 1992; Jaffe et al., Nature Genetics, 1, 372-378, 1992). We have used a defective recombinant adenovirus, Ad.RSV beta gal, containing the Escherichia coli beta-galactosidase gene targeted to the nucleus under the transcriptional control of the Rous sarcoma virus long terminal repeat promoter (Stratford-Perricaudet et al., J. Clin. Invest., 90, 626-630, 1992) to infect non-dividing neural cells in primary culture. We show that 80-100% of neuronal and astroglial cells infected with a viral titre lower than 10(9) p.f.u./ml express beta-galactosidase for at least 1 month without cell damage. These results demonstrate the potential usefulness of recombinant adenovirus infection for the analysis of brain-specific gene regulation and for the transfer of genes into neural cells before their transplantation into the brain.