Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 18 de 18
Filtrar
1.
Gene Ther ; 19(1): 15-24, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21562592

RESUMO

Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.


Assuntos
Técnicas de Transferência de Genes , Vetores Genéticos/metabolismo , Elementos Isolantes , Fatores de Transcrição NFI/metabolismo , Animais , Sítios de Ligação , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Fator de Ligação a CCCTC , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Vírus da Leucemia Murina de Friend/genética , Vírus da Leucemia Murina de Friend/metabolismo , Inativação Gênica , Vetores Genéticos/genética , Células HeLa , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFI/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Proto-Oncogenes/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Transfecção , Transgenes , Integração Viral
2.
Hum Mol Genet ; 10(23): 2717-26, 2001 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-11726559

RESUMO

The autosomal dominant mutation causing myotonic dystrophy (DM1) is a CTG repeat expansion in the 3'-UTR of the DM protein kinase (DMPK) gene. This multisystemic disorder includes myotonia, progressive weakness and wasting of skeletal muscle and extramuscular symptoms such as cataracts, testicular atrophy, endocrine and cognitive dysfunction. The mechanisms underlying its pathogenesis are complex. Recent reports have revealed that DMPK gene haploinsufficiency may account for cardiac conduction defects whereas cataracts may be due to haploinsufficiency of the neighboring gene, the DM-associated homeobox protein (DMAHP or SIX5) gene. Furthermore, mice expressing the CUG expansion in an unrelated mRNA develop myotonia and myopathy, consistent with an RNA gain of function. We demonstrated that transgenic mice carrying the CTG expansion in its human DM1 context (>45 kb) and producing abnormal DMPK mRNA with at least 300 CUG repeats, displayed clinical, histological, molecular and electrophysiological abnormalities in skeletal muscle consistent with those observed in DM1 patients. Like DM1 patients, these transgenic mice show abnormal tau expression in the brain. These results provide further evidence for the RNA trans-dominant effect of the CUG expansion, not only in muscle, but also in brain.


Assuntos
Encéfalo/anormalidades , Músculo Esquelético/anormalidades , Proteínas Serina-Treonina Quinases/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Eletromiografia , Eletroforese em Gel de Poliacrilamida , Feminino , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/citologia , Miotonia/genética , Miotonia/fisiopatologia , Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Miotonina Proteína Quinase , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Repetições de Trinucleotídeos/genética , Proteínas tau/metabolismo
3.
Hum Mol Genet ; 9(8): 1185-94, 2000 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-10767343

RESUMO

Myotonic dystrophy (DM) is caused by a CTG repeat expansion in the 3'UTR of the DM protein kinase (DMPK) gene. A very high level of instability is observed through successive generations and the size of the repeat is generally correlated with the severity of the disease and with age at onset. Furthermore, tissues from DM patients exhibit somatic mosaicism that increases with age. We generated transgenic mice carrying large human genomic sequences with 20, 55 or >300 CTG, cloned from patients from the same affected DM family. Using large human flanking sequences and a large amplification, we demonstrate that the intergenerational CTG repeat instability is reproduced in mice, with a strong bias towards expansions and with the same sex- and size-dependent characteristics as in humans. Moreover, a high level of instability, increasing with age, can be observed in tissues and in sperm. Although we did not observe dramatic expansions (or 'big jumps' over several hundred CTG repeats) as in congenital forms of DM, our model carrying >300 CTG is the first to show instability so close to the human DM situation. Our three models carrying different sizes of CTG repeat provide insight on the different factors modulating the CTG repeat instability.


Assuntos
Distrofia Miotônica/genética , Proteínas Serina-Treonina Quinases/genética , Repetições de Trinucleotídeos , Regiões 3' não Traduzidas/genética , Animais , Clonagem Molecular , Feminino , Biblioteca Gênica , Impressão Genômica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Miotonina Proteína Quinase , Proteínas Recombinantes/biossíntese , Espermatozoides/fisiologia
4.
Hum Mol Genet ; 7(8): 1285-91, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9668171

RESUMO

A (CTG)nexpansion in the 3'-untranslated region (UTR) of the DM protein kinase gene ( DMPK ) is responsible for causing myotonic dystrophy (DM). Major instability, with very large expansions between generations and high levels of somatic mosaicism, is observed in patients. There is a good correlation between repeat size (at least in leucocytes), clinical severity and age of onset. The trinucleotide repeat instability mechanisms involved in DM and other human genetic diseases are unknown. We studied somatic instability by measuring the CTG repeat length at several ages in various tissues of transgenic mice carrying a (CTG)55expansion surrounded by 45 kb of the human DM region, using small-pool PCR. These mice have been shown to reproduce the intergenerational and somatic instability of the 55 CTG repeat suggesting that surrounding sequences and the chromatin environment are involved in instability mechanisms. As observed in some of the tissues of DM patients, there is a tendency for repeat length and somatic mosaicism to increase with the age of the mouse. Furthermore, we observed no correlation between the somatic mutation rate and tissue proliferation capacity. The somatic mutation rates in different tissues were also not correlated to the relative inter-tissue difference in transcriptional levels of the three genes (DMAHP , DMPK and 59) surrounding the repeat.


Assuntos
Distrofia Miotônica/genética , Distrofia Miotônica/patologia , Distrofia Miotônica/fisiopatologia , Proteínas Serina-Treonina Quinases/genética , Sequências Repetitivas de Ácido Nucleico , Transcrição Gênica , Fatores Etários , Animais , Divisão Celular , Humanos , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Miotonina Proteína Quinase
5.
Nat Genet ; 15(2): 190-2, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9020847

RESUMO

Myotonic dystrophy (DM) is associated with the expansion of a (CTG)n trinucleotide repeat in the 3' untranslated region (UTR) of the DM protein kinase gene (DMPK). The (CTG)n repeat is polymorphic and varies in size between 5 and 37 repeats in unaffected individuals whereas in affected patients there are between 50 and 4,000 CTGs. The size of the (CTG)n repeat, which increases through generations, generally correlates with clinical severity and age of onset. The instability of the CTG repeat appears to depend on its size as well as on the sex of the transmitting parent. Moreover, mitotic instability analysis of different human DM tissues shows length mosaicism between different cell lineages. The molecular mechanisms of triplet instability remain elusive. To investigate the role of genomic sequences in instability, we produced transgenic mice containing a 45-kb genomic segment with a 55-CTG repeat cloned from a mildly affected patient. In contrast to other mouse models containing CAG repeats within cDNAs, these mice showed both intergenerational and somatic repeat instability.


Assuntos
Distrofia Miotônica/genética , Transgenes/genética , Repetições de Trinucleotídeos , Animais , Cosmídeos/genética , DNA Complementar/genética , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mosaicismo , Mutação , Reação em Cadeia da Polimerase
6.
J Med Genet ; 31(1): 33-6, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8151634

RESUMO

The molecular basis of myotonic dystrophy (DM) has been characterised. All DM mutations characterised to date appear as an unstable elongation of a fragment containing a tandem repeat of a CTG motif, which can be visualised in both EcoRI and BamHI digests. It has been shown that the fragment is polymorphic in the normal population. Another 1 kb insertion/deletion polymorphism located near the unstable CTG repeat region has been identified. The 1 kb insertion allele is present in all DM patients. These different polymorphic systems can be distinguished using cDNA25 and BamHI, because this enzyme cuts between the site of the 1 kb insertion and the CTG repeat. We thus haplotyped DM patients from 72 French families and clearly showed that all chromosomes (100%) with the DM mutation carried the 1 kb insertion as well. In addition to this association, we detected significant linkage disequilibrium between the DM locus and D19S63 for which allelic frequencies were different from other European populations. Our results in the French DM population are thus in agreement with the hypothesis that the CTG expansion occurred on one or a few ancestral chromosomes carrying the large 1 kb insertion allele.


Assuntos
Aberrações Cromossômicas , Desequilíbrio de Ligação , Distrofia Miotônica/genética , Sequências Repetitivas de Ácido Nucleico , Alelos , Sondas de DNA , Frequência do Gene , Haplótipos , Humanos , Mutação , Mapeamento por Restrição
7.
Hum Mol Genet ; 2(8): 1263-6, 1993 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7691346

RESUMO

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease. The mutation has been identified as an unstable trinucleotide CTG repeat in a sequence encoding a putative cAMP-dependent protein kinase. The CTG repeat varies in length between affected siblings, and generally increases through generations in parallel with increasing severity of the disease. Congenital myotonic dystrophy, which represents the most severe phenotype, is exclusively maternally inherited. In this report, we show, by Northern blot analysis, that no mutated enlarged transcript is detectable in a 20-week-old DM fetus and in two congenitally affected infants. Furthermore, in skeletal and cardiac muscle of the DM fetus, we observed by RNA analysis, including Northern blot and RT-PCR, an unexpectedly low expression of the paternal wild type allele. Varying degrees of expression of the mutant and/or the normal allele might therefore account for the characteristic features of the congenital form and the extreme variability of the disease.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Expressão Gênica , Distrofia Miotônica/genética , Sequências Repetitivas de Ácido Nucleico , Alelos , Sequência de Bases , Northern Blotting , Southern Blotting , DNA/análise , Primers do DNA , Genes Dominantes , Humanos , Recém-Nascido , Dados de Sequência Molecular , Músculos/enzimologia , Mutação , Miocárdio/enzimologia , Distrofia Miotônica/congênito , Reação em Cadeia da Polimerase/métodos , RNA/análise , Transcrição Gênica
8.
Am J Hum Genet ; 52(5): 875-83, 1993 May.
Artigo em Inglês | MEDLINE | ID: mdl-8098180

RESUMO

Myotonic dystrophy (DM) is a progressive neuromuscular disorder which results from elongations of an unstable (CTG)n repeat, located in the 3' untranslated region of the DM gene. A correlation has been demonstrated between the increase in the repeat number of this sequence and the severity of the disease. However, the clinical status of patients cannot be unambiguously ascertained solely on the basis of the number of CTG repeats. Moreover, the exclusive maternal inheritance of the congenital form remains unexplained. Our observation of differently sized repeats in various DM tissues from the same individual may explain why the size of the mutation observed in lymphocytes does not necessarily correlate with the severity and nature of symptoms. Through a molecular and genetic study of 142 families including 418 DM patients, we have investigated the dynamics of the CTG repeat meiotic instability. A positive correlation between the size of the repeat and the intergenerational enlargement was observed similarly through male and female meioses for < or = 0.5-kb CTG sequences. Beyond 0.5 kb, the intergenerational variation was more important through female meioses, whereas a tendency to compression was observed almost exclusively in male meioses, for > or = 1.5-kb fragments. This implies a size- and sex-dependent meiotic instability. Moreover, segregation analysis supports the hypothesis of a maternal as well as a familial predisposition for the occurrence of the congenital form. Finally, this analysis reveals a significant excess of transmitting grandfathers partially accounted for by increased fertility in affected males.


Assuntos
Meiose/genética , Mosaicismo , Distrofia Miotônica/genética , Sequências Repetitivas de Ácido Nucleico , Adulto , Criança , Análise Mutacional de DNA , Pai , Feminino , Variação Genética , Humanos , Masculino , Mães , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Recombinação Genética
9.
J Med Genet ; 28(2): 89-91, 1991 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-2002492

RESUMO

We report on two cases of prenatal diagnosis of myotonic dystrophy (DM), using flanking markers APOC2 or CKMM on the proximal side and D19S51 on the distal side. By double digestion (TaqI and NcoI) of PCR amplified CKMM, the informativeness was increased from a PIC value of 0.57 to 0.69. Altogether, with a PIC value of 0.64 for APOC2, 0.69 for CKMM, and 0.27 for D19S51 (BglI), presymptomatic and prenatal diagnosis can thus be offered to approximately 24% of persons with a risk between 0.0004 and 0.0008 using these flanking markers.


Assuntos
Cromossomos Humanos Par 19 , Marcadores Genéticos , Distrofia Miotônica/diagnóstico , Distrofia Miotônica/genética , Diagnóstico Pré-Natal , Adulto , Sequência de Bases , Amostra da Vilosidade Coriônica , Mapeamento Cromossômico , Feminino , Ligação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Gravidez
11.
Ann Genet ; 30(1): 5-16, 1987.
Artigo em Francês | MEDLINE | ID: mdl-3498430

RESUMO

Carrier diagnosis and prenatal diagnosis of Duchenne's muscular dystrophy (DMD) and Becker's muscular dystrophy (BMD) has become possible using some twenty RFLPs detected by more than a dozen Xp21 probes that are either intragenic or flanking the disease locus. Results from familial studies on 88 DMD and BM families stress important considerations concerning a priori and final risks, individuals necessary for the identification of the phase, and the different strategies that can be applied, regardless of whether the study concerns an on-going pregnancy or a carrier-status determination, and whether the patient is at high or low risk. Finally, multiple sources of difficulties in interpreting the results depend on a) the occurrence of new mutations that must be traced; b) the existence of meiotic recombination; c) the necessity, in some instances, of relying upon the sole identification of the paternal X. These considerations emphasize the characteristics and the important limitations of this type of methodology.


Assuntos
Creatina Quinase/genética , Distrofias Musculares/diagnóstico , Feminino , Triagem de Portadores Genéticos , Humanos , Masculino , Modelos Genéticos , Distrofias Musculares/classificação , Distrofias Musculares/genética , Linhagem , Polimorfismo Genético , Gravidez , Diagnóstico Pré-Natal , Software
12.
Nature ; 322(6074): 73-7, 1986.
Artigo em Inglês | MEDLINE | ID: mdl-3014348

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder for which the biochemical defect is as yet unknown. Recently, two cloned segments of human X-chromosome DNA have been described which detect structural alterations within or near the genetic locus responsible for the disorder. Both of these cloned segments were described as tightly linked to the locus and were capable of detecting deletions in the DNA of boys affected with DMD. In an attempt to determine more precisely the occurrence of these deletions within a large population of DMD patients and the accuracy of one of the segments, DXS164 (pERT87), in determining the inheritance of the DMD X chromosome, the subclones 1, 8 and 15 were made available to many investigators throughout the world. Here we describe the combined results of more than 20 research laboratories with respect to the occurrence of deletions at the DXS164 locus in DNA samples isolated from patients with DMD and Becker muscular dystrophy (BMD). The results indicate that the DXS164 locus apparently recombines with DMD 5% of the time, but is probably located between independent sites of mutation which yield DMD. The breakpoints of some deletions are delineated within the DXS164 locus, and it is evident that the deletions at the DMD locus are frequent and extremely large.


Assuntos
Deleção Cromossômica , DNA/análise , Desoxirribonucleases de Sítio Específico do Tipo II , Distrofias Musculares/genética , Mapeamento Cromossômico , Enzimas de Restrição do DNA/metabolismo , Desoxirribonuclease EcoRI , Eletroforese em Gel de Poliacrilamida , Genes , Humanos , Masculino , Linhagem
13.
Sem Hop ; 59(44): 3038-41, 1983 Dec 01.
Artigo em Francês | MEDLINE | ID: mdl-6320390

RESUMO

A prospective serologic study was conducted to determine the incidence of primary Cytomegalovirus (CMV) infection after a renal transplant and to evaluate the consequences on the graft. Serologic tests using the indirect hemagglutination technique with CMV-sensitized erythrocytes were performed serially over 6 years in 370 children initially awaiting renal transplantation. 211 children received a graft during the study period. Transplantation was followed by primary CMV infection in 48 (35.3%) of the 136 children with negative serologic tests at the time of the procedure. The percentage of graft failures is identical in the 75 children with positive tests before transplantation and in the 88 children with negative tests before and after transplantation (24 and 23% respectively). Conversely, only 6 (12.5%) graft failures were recorded among the 48 children who experienced primary CMV infection after transplantation. It can be speculated that these results are due to the immunosuppressive effect of CMV infection.


Assuntos
Infecções por Citomegalovirus/epidemiologia , Transplante de Rim , Adolescente , Adulto , Criança , Pré-Escolar , Infecções por Citomegalovirus/imunologia , Sobrevivência de Enxerto , Humanos , Estudos Prospectivos , Testes Sorológicos
15.
J Clin Microbiol ; 13(6): 1026-30, 1981 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-6265494

RESUMO

An indirect hemagglutination test with lyophilized, fixed, tanned, and cytomegalovirus (CMV)-sensitized sheep erythrocytes for the detection of CMV antibodies is reported. To avoid nonspecific hemagglutination, cells were fixed with glutaraldehyde or Formalin directly in whole blood. The lyophilized, CMV-sensitized erythrocytes obtained by this technique were stable up to 9 months at 37 degrees C and retained the same reactivity at fresh, CMV-sensitized cells. Indirect hemagglutination performed with lyophilized, sensitized cells was highly efficient in detecting CMV-antibodies as compared with complement fixation and enzyme immunoassay.


Assuntos
Anticorpos Antivirais/análise , Citomegalovirus/imunologia , Testes de Hemaglutinação/métodos , Animais , Testes de Fixação de Complemento , Eritrócitos/imunologia , Liofilização , Humanos , Técnicas Imunoenzimáticas , Ovinos/imunologia
16.
Arch Fr Pediatr ; 37 Suppl 2: I-IV, 1980.
Artigo em Francês | MEDLINE | ID: mdl-6255886

RESUMO

Two hundred children and their mothers were examined when they were 10 months, 2 years and 4 years of age. Anti-cytomegalovirus, anti-Herpesvirus hominis and anti-rubella virus antibodies were titered. The evolution of antibody titers showed that primary infections with cytomegalovirus and Herpesvirus are especially frequent during the first months of life, and that mothers are the principal source of infection. In the group studied, it appeared that country of origin of the parents was the most important socio-economic factor influencing the frequency of infections in children, based upon the large variations of the serum positivity in mothers.


Assuntos
Infecções por Citomegalovirus/epidemiologia , Herpes Simples/epidemiologia , Argélia/etnologia , Anticorpos Antivirais/análise , Pré-Escolar , Infecções por Citomegalovirus/transmissão , Feminino , Herpes Simples/transmissão , Humanos , Lactente , Estudos Longitudinais , Paris , Portugal/etnologia , Gravidez , Testes Sorológicos , Fatores Socioeconômicos , Espanha/etnologia
17.
Artigo em Francês | MEDLINE | ID: mdl-409756

RESUMO

The different results that have been published concerning the problem of the final outcome for patients who have had influenza during pregnancy reported in numerous works might be explained by differences in methodology used by the different authors. A study was carried out in the Haguenau Maternity Hospital (Bas-Rhin) when an epidemic of influenza occurred in 1972-173. The virological diagnosis was made by carrying out separate serological estimations on 1940 pregnant women. It can be shown that the mean birthweight dropped in infants of mothers who had contracted influenza during pregnancy even when corrections were made for sex and the duration of the pregnancy. The drop in weight of the placenta (37.3 g as a mean) was more obvious and can totally explain the drop in fetal weight. It was not possible to demonstrate any increase in the number of congenital malformations that were found in the neonatal period in the infants born to women who had had influenza. These results suggest that there is no direct passage of the influenza virus across the placenta which, however, is itself modified by the infection.


Assuntos
Peso ao Nascer , Influenza Humana , Placenta , Complicações Infecciosas na Gravidez , Animais , Embrião de Galinha , Feminino , Haplorrinos , Humanos , Influenza Humana/etiologia , Tamanho do Órgão , Gravidez
18.
Pathol Biol (Paris) ; 24(8): 575-9, 1976 Oct.
Artigo em Francês | MEDLINE | ID: mdl-185575

RESUMO

Indirect hemagglutination test for detection of antibodies to cytomegalovirus is highly sensitive and reproducible, if employed in well-defined conditions. Standardization of the various factors involved is necessary as well as their reciprocal equilibrium : sheep erythrocytes, antigen, dilution of tanin, buffers quality. The hemagglutination test can be performed on small volumes such as blood collection on blotting paper (PKU). Antibody titers were compared in the serum and the blood so collected in 104 subjects : the results were very similar and no "false negative" were found in any case. This way of collecting blood and hemagglutination are technical improvements in epidemiologic studies of cytomegalovirus infection. It can be hoped they will be adapted to other group herpes infections.


Assuntos
Anticorpos Antivirais/análise , Citomegalovirus/imunologia , Testes de Hemaglutinação/métodos , Animais , Coleta de Amostras Sanguíneas/métodos , Soluções Tampão , Infecções por Citomegalovirus/diagnóstico , Humanos , Ovinos/imunologia , Taninos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...