Asunto(s)
Proteínas de Homeodominio/genética , Hipoventilación/genética , Mutación , Péptidos/genética , Trastornos Intrínsecos del Sueño/genética , Factores de Transcripción/genética , Alelos , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Análisis Mutacional de ADN , Expansión de las Repeticiones de ADN , Secuencia Rica en GC , Heterocigoto , Humanos , Hipoventilación/congénito , Hipoventilación/diagnóstico , Recién Nacido , Datos de Secuencia Molecular , Fenotipo , Reacción en Cadena de la Polimerasa , Trastornos Intrínsecos del Sueño/congénito , Trastornos Intrínsecos del Sueño/diagnóstico , SíndromeRESUMEN
WAG/Rij rat strain has been suggested as an animal model for the study of inherited human medullary thyroid carcinoma (MTC), due to its high incidence of spontaneous C-cell thyroid tumours. Although the role of the Ret proto-oncogene mutations, as responsible for human MTC, is well established, nothing has been published concerning this putative animal model. Based upon the previously reported rat Ret sequence, exons 10, 11, 13, 14, 15, and 16, known to carry activating mutations in humans, have been analysed in the WAG/Rij rat by PCR, single strand conformational polymorphism (SSCP) and direct sequencing. Neither the germline nor MTC samples showed any Ret sequence difference in the exons when analysed in comparison to a non-MTC-susceptible rat strain. Our results indicate that Ret exons relevant in humans are not involved in WAG/Rij rat MTC, as expected, and this questions the validity of this strain as a model for the human disease, and suggests there must be additional mechanisms for the genesis and progression of rat MTC.
Asunto(s)
Carcinoma Medular/genética , Modelos Animales de Enfermedad , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Neoplasias de la Tiroides/genética , Animales , Exones , Femenino , Mutación de Línea Germinal , Humanos , Masculino , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret , Ratas , Ratas Mutantes , Ratas Sprague-Dawley , Alineación de SecuenciaRESUMEN
The RET proto-oncogene, a member of the Receptor Tyrosine Kinase family, plays a crucial role during the development of the excretory system and the enteric nervous system, as demonstrated by in vivo animal studies and by its involvement in the pathogenesis of several human neurocristopathies like Hirschsprung disease and Multiple Endocrine Neoplasia type 2. Using a multistep RT-PCR approach we have isolated and sequenced the cDNA of the whole rat RET proto-oncogene, reporting the deduced amino acid sequence in comparison with the human and mouse counterparts. Moreover, two different isoforms (RET9 and RET51) have been confirmed in the rat, while a third RET isoform demonstrated in human (RET43) has not resulted to be conserved in this species. Finally, we have determined the genomic structure of the rat RET proto-oncogene comparing the exon-intron boundaries and intron sizes with the known structure of the human homologous gene. Our findings will facilitate the molecular study of appropriate rat models of RET related human diseases.
Asunto(s)
Proteínas de Drosophila , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Proteínas Tirosina Quinasas Receptoras/genética , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Artificiales de Levadura , ADN Complementario , Exones , Genoma Humano , Humanos , Intrones , Ratones , Datos de Secuencia Molecular , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic Medullary Thyroid Carcinoma (MTC) cases. They are represented by the Met918Thr substitution (exon 16) typical of Multiple Endocrine Neoplasia type 2B (MEN2B) and, to a lesser extent, by nucleotide changes occurring at one of five critical cysteine residues (exons 10 and 11) typical of MEN type 2A (MEN2A). An in vitro transforming activity has already been demonstrated for these mutations. A few different MTC somatic mutations have been reported so far whose biological activity has still to be tested. In this paper we report the identification, in two MTC tumor samples, of two interstitial deletions of 48 bp and 6 bp occurred in exons 10 and 11 respectively. Both were somatic heterozygous in frame mutations, not involving any cysteine residue. Moreover, the expression of a full length RET cDNA carrying one of the two deletions demonstrated a strong transforming capacity in NIH3T3 cells.
Asunto(s)
Cisteína , Proteínas de Drosophila , Regulación Neoplásica de la Expresión Génica , Proteínas Proto-Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Eliminación de Secuencia , Células 3T3 , Adulto , Animales , Carcinoma Medular/genética , Transformación Celular Neoplásica , Femenino , Humanos , Masculino , Ratones , Polimorfismo Conformacional Retorcido-Simple , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret , Neoplasias de la Tiroides/genética , TransfecciónAsunto(s)
Mapeo Cromosómico , Proteínas de Drosophila , Proteínas Proto-Oncogénicas/genética , Ratas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ligamiento Genético , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas c-ret , Ratas Endogámicas , Homología de Secuencia de AminoácidoRESUMEN
Tight linkage with the RET proto-oncogene (Zmax = 3.41 at theta = 0.00), analysis of recombinants and detection of a familial microdeletion in a large pedigree restrict the mapping of the Hirschsprung (HSCR) gene previously localized on proximal 10q. The molecular characterization of the familial microdeletion and of 3 additional cytogenetically visible de novo deletions, isolated in somatic cell hybrids, identify a smallest region of overlap of 250 Kb. This contains the RET proto-oncogene where missense mutations causing multiple endocrine neoplasia type 2A (MEN 2A) phenotype were recently found. The pentagastrin test (which detects preclinical forms of MEN 2A or B) is negative in adult HSCR patients with deletions of the RET gene. This represents a good candidate for the search of mutations causing HSCR.
Asunto(s)
Cromosomas Humanos Par 10 , Proteínas de Drosophila , Eliminación de Gen , Enfermedad de Hirschsprung/genética , Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes , Proteínas Tirosina Quinasas Receptoras/genética , Línea Celular , Mapeo Cromosómico , Femenino , Genes Dominantes , Ligamiento Genético , Marcadores Genéticos , Humanos , Células Híbridas , Hibridación Fluorescente in Situ , Masculino , Neoplasia Endocrina Múltiple/genética , Linaje , Proteínas Tirosina Quinasas/genética , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-retRESUMEN
Three hundred fifteen radiation hybrids (RH) were isolated using a monochromosomal cell hybrid containing chromosome 16 only. A panel of 18 RH, which showed breakpoints among four markers (3.15, 26.6, 3'HVR, and 5'HVR) mapping in the distal portion of 16p, were selected and characterized for the retention of nine additional DNA sequences already localized in this region, and for one centromeric sequence. One or more breakpoints were identified in nine of the 12 intervals defined by the 13 single-copy sequences used. This panel of RH represents a tool for the construction of a detailed physical map of the distal part of 16p and for cloning sequences located in the proximity of disease genes. Three inter-Alu DNA sequences, amplified from one of these RH containing the autosomal dominant polycystic kidney disease (PKD1) gene, were cloned and mapped in the panel. Sequencing of the ends of one of three clones showed a (CAAA)n repeat, which revealed a two-allele polymorphism.