RESUMEN
We investigated the possible role of chromosome 10q losses in colorectal cancer metastasis by carrying out an allelic imbalance study on a series of microsatellite instability-negative (MSI-) primary tumours (n=32) and metastases (n=36) from 49 patients. Our results demonstrate that 10q allelic losses are associated with a significant proportion (25%) of MSI- colorectal tumours, but are not involved in the metastatic process. PTEN and BMPR1A, two genes located in the common deleted region, were screened for mutations in samples with loss of heterozygosity. The absence or low frequency of mutations indicates that the inactivation of these genes by deletion of one allele and mutation of the other one plays only a minor role in MSI- tumours.
Asunto(s)
Cromosomas Humanos Par 10 , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Pérdida de Heterocigocidad , Metástasis de la Neoplasia , Monoéster Fosfórico Hidrolasas/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento/genética , Proteínas Supresoras de Tumor/genética , Secuencia de Bases , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1 , Genes Supresores de Tumor , Mutación de Línea Germinal , Humanos , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Fosfohidrolasa PTENRESUMEN
Germline specific point mutations in the gene encoding fibroblast growth factor receptor 3 (FGFR3) are associated with autosomal dominant human skeletal dysplasia and craniosynostosis syndromes. Mutations identical to the germinal activating mutations found in severe skeletal dysplasias have been identified in certain types of cancer: at low frequency in multiple myeloma and cervix carcinoma and at high frequency in bladder carcinoma. We analysed, by SSCP and sequencing, the prevalence of FGFR3 mutations in 116 primary tumours of various types (upper aerodigestive tract, oesophagus, stomach, lung and skin). The regions analysed encompassed all FGFR3 point mutations previously described in severe skeletal dysplasia and cancers. No mutations were detected in the tumour types examined, suggesting that FGFR3 mutations are restricted to a few tumour types, the evidence to date suggesting that they are very specific to bladder carcinomas.
Asunto(s)
Carcinoma/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Neoplasias de la Vejiga Urinaria/genética , Enfermedades del Desarrollo Óseo/genética , Humanos , Oncogenes , Mutación Puntual , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
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.
Asunto(s)
Distrofia Miotónica/genética , Proteínas Serina-Treonina Quinasas/genética , Repeticiones de Trinucleótidos , Regiones no Traducidas 3'/genética , Animales , Clonación Molecular , Femenino , Biblioteca de Genes , Impresión Genómica , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteína Quinasa de Distrofia Miotónica , Proteínas Recombinantes/biosíntesis , Espermatozoides/fisiologíaRESUMEN
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.
Asunto(s)
Distrofia Miotónica/genética , Distrofia Miotónica/patología , Distrofia Miotónica/fisiopatología , Proteínas Serina-Treonina Quinasas/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Factores de Edad , Animales , División Celular , Humanos , Ratones , Ratones Transgénicos , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Proteína Quinasa de Distrofia MiotónicaRESUMEN
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.
Asunto(s)
Distrofia Miotónica/genética , Transgenes/genética , Repeticiones de Trinucleótidos , Animales , Cósmidos/genética , ADN Complementario/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Mosaicismo , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
Expansion of (C+G)-rich trinucleotide repeats has been shown to be associated with several autosomal or X-linked genetic diseases and/or fragile sites. By analysing the sequences available in the databases, we found, in a significant proportion of triplet associated genes or fragile sites (11/12), a CpG island close to the trinucleotide repeat. This association led us to assume that flanking regions and chromatin structure near the triplets might play a role in repeat instability.
Asunto(s)
Aberraciones Cromosómicas/genética , Islas de CpG , Aberraciones Cromosómicas Sexuales/genética , Repeticiones de Trinucleótidos , Cromosoma X , Trastornos de los Cromosomas , Ligamiento Genético , HumanosRESUMEN
The mutation causing myotonic dystrophy (DM) is an unstable CTG trinucleotide repeat in a gene encoding for a protein with putative serine-threonine kinase activity. Several studies have reported the appearance of abnormally frequent neurofibrillary tangles (NFTs) in the cortex of patients with DM. Using immunologic probes against normal and pathologic hyperphosphorylated tau proteins, the basic components of NFTs, we performed a biochemical and immunohistochemical study of the brains of two DM cases. We compared the tau profiles with those found in Alzheimer's disease (AD) using mono- and two-dimensional immunoblotting. Patients were aged 53 and 61 years at death. In both cases, we observed few perikaryal and axonal inclusions in the hippocampus as well as the entorhinal and inferior temporal cortices. As in AD brain homogenates, pathologic tau proteins, named tau 55, 64, and 69, were exclusively immunodetected in the DM cases in the hippocampus, the entorhinal cortex, and in most of the temporal areas. Amounts of pathologic tau proteins were higher in the more severely affected case, but lower than in AD brain homogenates. Pathologic tau proteins were less acidic in DM than in AD. We found a very low amount of the tau 69 isoform in DM extracts, and in most of the cortical areas, tau 55 was overexpressed compared with AD homogenates. A link between the increase of kinase activity and the presence of pathologic tau proteins is discussed.
Asunto(s)
Encéfalo/metabolismo , Distrofia Miotónica/metabolismo , Proteínas tau/análisis , Humanos , Immunoblotting , Inmunohistoquímica , Masculino , Persona de Mediana EdadRESUMEN
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.
Asunto(s)
Aberraciones Cromosómicas , Desequilibrio de Ligamiento , Distrofia Miotónica/genética , Secuencias Repetitivas de Ácidos Nucleicos , Alelos , Sondas de ADN , Frecuencia de los Genes , Haplotipos , Humanos , Mutación , Mapeo RestrictivoRESUMEN
Myotonic dystrophy (DM) results from the amplification of an unstable (CTG)n sequence in the 3' untranslated region of the myotonin-protein kinase (MT-PK) gene. The expression of the enlarged allele in DM patients with a number of repeats below or beyond 200, was analysed by three different groups. Two groups showed a decreased or absent expression of mutant alleles in DM adults, in congenitally affected infants (CDM) and in an affected fetus. On the contrary, another group reported the increased expression of the mutated allele in several tissues of a CDM infant. These discrepancies may be explained by the different methods used, the small number of patients, the individuals and tissues used as controls, or reflect the use of primers located in different regions of the MT-PK gene.
Asunto(s)
Expresión Génica , Distrofia Miotónica/genética , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas , Secuencias Repetitivas de Ácidos Nucleicos , Adulto , Secuencia de Bases , Feto , Humanos , Proteína Quinasa de Distrofia Miotónica , Especificidad de Órganos , Proteínas Quinasas/biosíntesis , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Mapeo RestrictivoRESUMEN
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.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/genética , Expresión Génica , Distrofia Miotónica/genética , Secuencias Repetitivas de Ácidos Nucleicos , Alelos , Secuencia de Bases , Northern Blotting , Southern Blotting , ADN/análisis , Cartilla de ADN , Genes Dominantes , Humanos , Recién Nacido , Datos de Secuencia Molecular , Músculos/enzimología , Mutación , Miocardio/enzimología , Distrofia Miotónica/congénito , Reacción en Cadena de la Polimerasa/métodos , ARN/análisis , Transcripción GenéticaRESUMEN
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.